\ 

r 


BIOLOGY 

LIBRARY 

G 


MICROORGANISMS 


INCLUDING 


BACTERIA  AND  PROTOZOA 


A  PRAITK'AL  MAM  AL  FOE  STUDENTS,  PHYSICIANS 
AND  HEALTH  OFFICERS 


BY 

WILLIAM  HALLOCK  PARK,  M.D. 

I'HOFKSSOH     OF     BACTERIOLOGY    AND    HYGIEXK,    UNIVERSITY'  'AND     BELLEVUE    HOSPITAL,    MEDICAL 

COLLEGE,    AND    DIRECTOR   OF   THE   RESEARCH    LABORATORY    OF   THE    DEPARTMENT 

OF    HEALTH,    CITY    OF    NEW    YORK 


ASSISTED  BY 

ANNA  W.  WILLIAMS,  M.D. 

ASSISTANT   DIRECTOR   OF  THE   RESEARCH   LABORATORY 

SECOND  EDITION,  ENLARGED  AND  THOROUGHLY  REVISED 
WITH    165  ENGRAVINGS  AND  4  FULL-PAGE  PLATES 


LEA   BROTHERS   &  CO. 

XKW   YORK   AND   PHILADELPHIA 
1905 


q 

BIOLOGY 

LIBRARY 

G 


Entered  according  to  the  Act  of  Congress,  in  the  year  1905,  by 

LEA   BROTHERS   &   CO., 
in  the  Office  of  the  Librarian  of  Congress.     All  rights  reserved. 


DORNAN,    PRINT  KH 


PEEFACE  TO  SECOND  EDITION 


THE  past  few  years  have  added  greatly  to  our  knowledge  of  the  two 
(lasses  of  pathogenic  micro-organisms,  bacteria  and  protozoa,  repre- 
M-nting  respectively  the  lowest  forms  of  the  vegetable  and  animal  king- 
doms. The  importance  of  the  protozoa  is  now  recognized,  not  only 
because  of  the  diseases  known  to  be  caused  by  them,  but  also  because 
of  their  possible  connection  with  the  exanthemata  and  syphilis.  In 
view  of  these  developments  it  is  obviously  essential  that  the  student 
should  be  instructed  in  both  fields.  I  believe  that  the  inclusion  of 
both  subjects  in  a  single  volume  offers  the  advantages  of  convenience, 
facility  of  instruction,  and  the  presentation  of  comprehensive  knowl- 
edge. In  the  present  edition  the  section  on  the  Protozoa,  excepting 
malaria,  was  undertaken  by  Dr.  A.  W.  Williams.  The  chapter  on 
Malaria  is  from  the  pen  of  Mr.  L.  B.  Goklhorn,  Instructor  in  Pathol- 
ogy in  the  University  and  Bellevue  Hospital  Medical  College.  Needless 
to  say,  the  revision  of  the  part  on  Bacteria  has  been  thorough,  so  that 
the  book  as  a  whole  endeavors  to  reflect  the  latest  knowledge  in  the 
whole  domain.  This  edition,  like  the  previous  one,  has  been  written  for 
the  student  and  physician  rather  than  for  the  laboratory  worker. 

In  the  preparation  of  this  edition  I  greatly  missed  the  active  assistance 
of  Dr.  Guerard,  whose  removal  to  a  distant  city  prevented  his  co-opera- 
tion, except  in  the  reading  of  the  proof.  I  am  also  indebted  to  Dr. 
Cliarles  Bolduan,  Assistant  Bacteriologist  in  the  Research  Laboratory, 
for  valuable  help  in  preparing  this  work  for  press. 

I  take  this  opportunity  of  acknowledging  much  valuable  help  from 
the  splendid  work  on  the  pathogenic  micro-organisms  edited  by  Kolle 
and  Wassermann.  Several  photographs  from  their  Atlas  have  been  used. 

W.  H.  P. 

XE\V  YORK,  1905. 


i  " 


CONTENTS. 


PART   I. 
PRINCIPLES  OF  BACTERIOLOGY. 


CHAPTER  I. 

PAGE 

Introductory— Historical  Sketch        .  .....       17 

CHAPTER  II. 

General  Characteristics  of   Bacteria — Relation  to   Other  Micro-organisms — 

Morphology  and  Structure  ........        23 

CHAPTER  III. 

The  Classification  of  Bacteria — Permanence  of  Species — Chemical  Composi- 
tion and  Nutrition  .........       36 

CHAPTER  IV. 
Effect  of  Temperature  upon  the  Growth  of  Bacteria     .  .       43 

CHAPTER  V. 
The  Materials  and  Methods  1'sed  in  the  Cultivation  of  Bacteria     ...       47 

CHAPTER  VI. 
Microscopic  Methods  .  68 

CHAPTER  VII. 

Vital  Phenomena  of  Barn-ria — Motion,  Heat,  and  Light  Production — Chem- 
ical  Effect  -  .        86 

CHAPTER  VIII. 
The  Effect  of  Various  Deleterious  Influences  upon  Bacteria       .  .      102 

CHAPTER  IX. 
The  Destruction  of  Bacteria  l>v  Chemicals — Practical  Use  of  Disinfectants    .      107 

CHAPTER   X. 

Practical    Disinfection   and   Sterilization  (House,   Person,   Instruments,  and 

I ••,„„!)—  Sterilization  of  Milk  for  Feeding  Infants 117 


vi  CONTENTS 

CHAPTER  XI. 

PAGE 

The  Use  of  Animals  for  Diagnostic  and  Test  Purposes       .  .135 

CHAPTER  XII. 

The  Procuring  of  Material  for  Bacteriological  Examination  from  Those  Suffer- 
ing from  Disease       .  .          .          .          .          .          .          .          .      138 

CHAPTER  XIII. 
The  Relation  of  Bacteria  to  Disease        .  .141 

CHAPTER  XIV. 

The  Antagonism  Existing  between  the  Living  Body  and  Micro-organisms      .      155 

CHAPTER  XV. 

Nature  of  the  Protective  Defences  of  the  Body  and  Their  Manner  of  Action 

— Ehrlich's  "Side-chain"  Theory        .  .      162 

CHAPTER  XVI. 
The  Nature  of  the  Substances  Concerned  in  Agglutination     .          .          .          .175 


PART   II. 

BACTERIA  PATHOGENIC  TO  MAN  INDIVIDUALLY 
CONSIDERED. 


CHAPTER  XVII. 
The  Bacillus  and  the  Bacteriology  of  Diphtheria       .          .          .          .          .185 

CHAPTER  XVIII. 

The  Bacillus  and  the  Bacteriology  of  Tetanus     .  .     222 

CHAPTER  XIX. 

The  Colon  Bacillus  Group,  Paracolon,  Paratyphoid  Dysentery  and  Paradysen- 

tery  Bacilli       .  ....  .234 

CHAPTER  XX. 
The  Typhoid  Bacillus  (Bacillus  Typhosus)        .  .     263 

CHAPTER  XXI. 

The  Bacillus  of  Tuberculosis  288 


CONTEXTS  vii 

CHAPTEB  XXII. 

PAGE 

Bacillus  Showing  Staining  Reactions  Similar  to  Those  of  the  Tubercle  Bacilli 
I.u<t«:artenV     Bacillus    -Sniegnia     Bacillus — Leprosy     Bacillus — Gra— 
Bacilli       .  .      316 

CHAPTER  XXIII. 
The  Influenza  and  Pseudoinfluenza  Bacilli — The  Koch-Weeks  Bacillus  .          .     321 

CHAPTKH   XXIV. 
The  Producers  of  Abscesses,  Cellulitis,  Septicaemia,  etc.       .  .     329 

CHAPTER  XXV. 

The  Diplococcus  of  Pneumonia  (Pneumococcus,  Streptococcus  Lanceolatus, 

Micrococcus  Lanceolatus) — The  Pneumobacillus  (Friedlander  Bacillus)    .     349 

CHAPTER   XXVI. 

Meningococcus  or  Diplococcus  (Micrococcus)  Intracellularis  Meningitidis,  and 

the   Relation  of  it  and  of  Other  Bacteria  to  Meningitis       .  .  .      360 

CHAPTER  XXVII. 

The  Gonococcus  or  Micrococcus  Gonorrhcese — The  Ducrey  Bacillus  of  Soft 

Chancre  .  .  .      366 

CHAPTER  XXVIII. 

Bacillus  Pyocyaneus  (Bacillus  of  Green  and  of  Blue  Pus) — Bacillus  Proteus 
Vulgaris — Group  of  Malignant  (Edema  Bacilli — Bacillus  Aerogenes  Cap- 
.sulatus  ............  374 

CHAPTER   XXIX. 
The  Anthrax   Bacillus  and  the  Bacillus  of  Symptomatic  Anthrax        .  .      382 

CHAPTER  XXX. 
The  Cholera  Spirillum  (Spirillum  Cholera'  Asiatic-re)  and  Allied  Varieties        .  "  393 

CHAPTER   XXXI. 
Clanders  Bacillus  (Bacillus  Mallei)     .  .  .      408 

CHAPTER  XXXII. 

Th<-  Bacillus  of  Bubonic  Plague — The  Bacillus  Icteroides — The  Micrococcus 

Melitensis  .  .     413 

CHAPTER  XXXIII. 

Representative  Pathogenic  Micro-organisms  Belonging  to  the  Higher  Bac- 
teria 418 


vijj  CONTENTS 

CHAPTER  XXXIV. 

PAGE 

The  Pathogenic  Fungi  and  Yeasts  (Blastomycetes) — Diseases  Due  to  Micro- 
organisms Not  yet  Identified      .  .     433 

CHAPTER  XXXV. 

The  Bacteriological  Examination  of  Water,  Air,  and  Soil — The  Contamina- 
tion and  Purification  of  Water — The  Disposal  of  Sewage      .  .     443 

CHAPTER  XXXVI. 

The  Bacteriology  of  Milk  in  its  Relation  to  Disease      .  ...     453 


PART   III. 

PROTOZOA. 


CHAPTER  XXXVII. 

Classification   and   General   Characteristics         .  .  .     469 

CHAPTER  XXXVIII. 
Amoebina         ..........  .     477 

CHAPTER  XXXIX. 

Trypanosoma  .  .  .  .  .  .  .  .  .    '       .  •      482 

CHAPTER  XL. 

Malarial  Parasitology        .....  .      500 

CHAPTER  XLI. 

Piroplasma  Bigeminum — The  Microsporidia — Balantidium  Coli     .  .  .514 

CHAPTER  XLII. 

Protozoan-like  Bodies  in  Smallpox  and  Allied  Diseases  (Cowpox,  Horsepox, 

Sheeppox),  and  in  Scarlet  Fever        .  .     521 

CHAPTER  XLIII. 
Kala-azar — Rabies  .  .  .  .  .  .  .  .          .  .      529 

APPENDIX. 

Aggressins — Experiments  Devised  by  Ehrlich  to  Show  the  Nature  of  Hamo- 
lysins — So-called  Ultrarnicroscopic  Examination — Variation  in  Suscep- 
tibility of  Guinea-pigs  to  Diphtheria  Toxin — Diplobacillus  of  Morax- 
Axenfeld — Bacteria  in  Ice — Stegomyia  Fasciata  and  its  Relation  to 
Yellow  Fever  .  539 


BACTERIOLOGY  IN  MEDICINE  AND  SURGERY. 


PART  I. 
PRINCIPLES  OF  BACTERIOLOGY. 


CHAPTER   I. 

INTRODUCTORY— HISTORICAL  SKETCH. 

ALTHOUGH  most  of  the  more  important  discoveries  in  bacteriology 
which  place  it  on  the  footing  of  a  science  are  of  comparatively  recent 
date,  the  foundations  of  its  study  were  laid  over  two  centuries  ago. 
From  the  earliest  times  the  history  of  bacteriology  has  been  intimately 
associated  with  that  of  medicine.  Indeed,  it  is  only  through  the  inves- 
tigations into  the  life  history  of  the  vegetable  and  animal  unicellular 
micro-organisms  that  our  present  knowledge  of  the  etiology,  course, 
and  prevention  of  the  infectious  diseases  has  been  acquired;  and  it  is 
only  by  the  practical  application  of  the  principles  and  methods  of 
bacteriology  that  many  diseases  can  be  positively  diagnosed  or  the 
problems  which  present  themselves  to  the  sanitarian  be  certainly  solved. 
The  prominent  position  which  bacteriology  already  holds  toward 
medicine  is,  moreover,  daily  increasing  in  importance.  Original  dis- 
coveries are  constantly  adding  to  the  list  of  known  germ  diseases,  and 
the  outlook  is  favorable  for  eventually  obtaining,  through  serums, 
through  attenuated  cultures,  or  through  the  toxic  substances  of  the 
micro-organisms  themselves,  means  for  immunizing  against,  if  not 
curing,  many  of  the  specific  infections.  Even  at  present  bacterial 
products  and  protective  serums  are  used  successfully  as  preventives  or 
curative  agents  in  several  of  the  most  prevalent  infectious  diseases. 
An  acquaintance,  therefore,  with  the  main  facts  concerning  these  micro- 
organisms is  as  necessary  to  the  education  of  the  modern  physician  as 
a  knowledge  of  anatomy,  pathology,  chemistry,  or  any  of  the  allied 
sciences. 

But  before  entering  into  a  detailed  consideration  of  the  subject  it 
may  be  interesting  and  instructive  to  review  briefly  a  few  of  the  impor- 

2 


18  PRINCIPLES  OF  BACTERIOLOGY 

tarit  steps  which  led  up  to  the  development  of  the  science,  and  upon 
which  ate  ^foundation  pests,an  which  we  shall  see  that  the  results  obtained 
\v<''-o  gained  only  .through1  long  and  laborious  research,  and  after  many 
obstacles  were  met  and  overcome  by  indomitable  perseverance  and 
accurate  observation  and  experiment. 

The  first  probably  authentic  observations  of  living  microscopic 
organisms  of  which  there  is  any  record  are  those  of  Kircher,  in  1659. 
This  original  investigator  demonstrated  the  presence  in  putrid  meat, 
milk,  vinegar,  cheese,  etc.,  of  "minute  living  worms,"  but  did  not 
describe  their  form  or  character. 

Not  long  after  this,  in  1675,  Leeuwenhoeck  observed  in  rain-water, 
putrid  infusions,  and  in  his  own  and  other  saliva  and  diarrhoeal  evacua- 
tions living,  motile  "  animalculse"  of  most  minute  dimensions,  which 
he  described  and  illustrated  by  drawings.  Leeuwenhoeck  practised  the 
art  of  lens-grinding,  in  which  he  eventually  became  so  proficient  that 
he  perfected  a  lens  superior  to  any  magnifying  glass  obtainable  at  that 
day,  and  with  which  he  was  enabled  to  see  objects  very  much  smaller 
than  had  ever  been  seen  before.  "With. the  greatest  astonishment,"  he 
writes,  "I  observed  distributed  everywhere  through  the  material  which 
I  was  examining  animalcules  of  the  most  microscopic  size,  which 
moved  themselves  about  very  energetically."  The  work  of  this  observer 
is  conspicuous  for  its  purely  objective  character  and  absence  of  specu- 
lation; and  his  descriptions  and  illustrations  are  done  with  remarkable 
clearness  and  accuracy,  considering  the  imperfect  optical  instruments 
at  his  command.  It  was  not  until  many  years  later,  however,  that  any 
attempt  was  made  to  define  the  characters  of  these  minute  organisms 
and  to  classify  them  systematically. 

From  the  earliest  investigations  into  the  life  history  and  properties 
of  bacteria  micro-organisms  have  been  thought  to  play  an  important 
part  in  the  causation  of  infectious  diseases.  Shortly  after  the  first 
investigations  into  this  subject  the  opinion  was  advanced  that  puerperal 
fever,  measles,  smallpox,  typhus,  pleurisy,  epilepsy,  gout,  and  many  other 
diseases  were  due  to  contagion.  In  fact,  so  widespread  became  the 
belief  in  a  causal  relation  of  these  minute  organisms  to  disease  that  it 
soon  amounted  to  a  veritable  craze,  and  all  forms  and  kinds  of  diseases 
were  said  to  be  produced  in  this  way,  upon  no  other  foundation  than 
that  these  organisms  had  been  found  in  the  mouth  and  intestinal  con- 
tents of  men  and  animals,  and  in  water. 

Among  those  who  were  especially  conspicuous  at  this  time  for  their 
advanced  views  on  the  germ-theory  of  infectious  diseases  was  Marcus 
Antonius  Plenciz,  a  physician  of  Vienna.  This  acute  observer,  who 
published  his  views  in  1762,  maintained  that  not  only  were  all  infectious 
diseases  caused  by  micro-organisms,  but  that  the  infective  material 
could  be  nothing  else  than  a  living  organism.  On  these  grounds  he 
endeavored  to  explain  the  variations  in  the  period  of  incubation  of  the 
different  infectious  diseases.  He  also  insisted  that  there  were  special 
germs  for  each  infectious  disease  by  which  the  specific  disease  was 
produced.  Plenciz  believed,  moreover,  that  these  organisms  were 


HISTORICAL  SKETCH  19 

capable  of  multiplication  in  the  body,  and  suggested  the  possibility  of 
their  being  conveyed  from  place  to  place  through  the  air. 

These  views,  it  is  true,  were  largely  speculative,  and  rested  upon 
insufficient  experiment;  but  they  were  so  plausible,  and  the  arguments 
put  forward  in  their  support  were  so  logical  and  convincing,  that  they 
continued  to  gain  ground,  in  spite  of  considerable  opposition  and 
ridicule,  and  in  many  instances  the  conclusions  reached  have  since 
been  proved  to  be  correct.  The  fact  that  infectious  diseases  were  of 
sudden  occurrence,  breaking  out  often  in  isolated  places,  and  that  they 
frequently  remained  clinging  for  long  periods  to  certain  localities, 
leaving  others  unaffected,  was  evidence  that  they  were  not  produced 
by  a  gaseous  infective  agent.  Moreover,  the  mode  of  infection,  its 
unlimited  development  among  large  numbers  of  individuals,  and 
gradual  spread  over  wide  areas — the  incubation,  course  of,  and  resulting 
immunity  in  recovery  from  infectious  diseases — all  pointed  to  the 
probable  cause  being  a  living  organism. 

Among  other  distinguished  men  of  the  day  whose  observations 
exerted  a  most  powerful  influence  upon  the  doctrine  of  infection  may 
be  mentioned  Henle.  His  writings  (Pathological  Investigations,  1840, 
and  Text-book  of  Rational  Pathology,  1853),  in  which  he  described  the 
relation  of  micro-organisms  to  infectious  diseases,  and  defined  the 
character  and  action  of  bacteria  upon  certain  phases  and  symptoms 
of  these  affections,  are  remarkable  for  their  clearness  and  precision. 

But,  meanwhile,  the  question  which  most  interested  these  investigators 
into  the  cause  of  infectious  diseases  was:  Whence  are  these  micro- 
organisms derived  which  were  supposed  to  produce  them?  Were  they 
the  result  of  spontaneous  generation  due  to  vegetative  changes  in  the 
substances  in  which  the  organisms  were  found,  or  were  they  reproduced 
from  similar  pre-existing  organisms — the  so-called  vitalistic  theory? 
This  question  is  intimately  connected  with  the  investigations  into  the 
origin  and  nature  of  fermentation  and  putrefaction. 

Spallanzani  in  1769  demonstrated  that  if  putrescible  infusions  of 
organic  matter  were  placed  in  hermetically  sealed  flasks  and  then 
boiled  the  liquids  were  sterilized;  neither  were  living  organisms  found 
in  the  solutions,  nor  did  they  decompose;  and  the  infusions  remained 
unchanged  for  an  indefinite  period. 

It  was  objected  to  these  experiments  that  the  high  temperature  to 
which  the  liquids  had  been  subjected  so  altered  them  that  spontaneous 
generation  could  no  longer  take  place.  This  objection  was  met  by 
Spallanzani  by  cracking  one  of  the  flasks  and. allowing  air  to  enter, 
when  living  organisms,  and  decomposition  again  appeared  in  the  boiled 
infusions. 

Another  objection  raised  by  the  believers  in  spontaneous  generation 
was  that  in  excluding  the  oxygen  of  the  air  by  hermetically  sealing  the 
flasks  the  essential  condition  for  the  development  of  fermentation,  which 
required  free  admission  of  this  gas,  was  interfered  with.  This  objection 
was  then  met  by  Schulze,  in  1836,  by  causing  the  air  admitted  to  the 
boiled  decomposable  liquids  to  pass  through  strong  sulphuric  acid. 


20  PRINCIPLES  OF  BACTERIOLOGY 

Air  thus  robbed  of  its  living  organisms  did  not  produce  decomposition; 
whereas  when  no  such  precautions  were  taken  with  the  air  admitted 
the  boiled  solutions  quickly  fell  into  putrefaction,  and  living  organisms 
were  found  to  be  present. 

Schwann  in  1839  obtained  similar  results  in  another  way;  he  deprived 
the  air  admitted  to  his  boiled  liquids  of  micro-organisms  by  passing  it 
through  a  tube  which  was  heated  to  a  temperature  high  enough  to 
destroy  them.  To  this  investigator  is  also  due  the  credit  of  having 
discovered  the  specific  cause — the  yeast  plant,  or  saccharomyces  cerevisice 
— of  alcoholic  fermentation,  the  process  by  which  sugar  is  decomposed 
into  alcohol  and  carbonic  acid. 

Again,  it  was  objected  to  these  experiments  that  the  heating  of  the 
air  had  perhaps  brought  about  some  chemical  change  which  hindered 
the  production  of  fermentation.  Schroeder  and  von  Dusch  in  1854 
then  showed  that  by  a  simple  process  of  filtration,  which  has  since 
proved  of  inestimable  value  in  bacteriological  work,  the  air  can  be 
mechanically  freed  from  germs.  By  placing  in  the  mouth  of  the  flask 
containing  the  boiled  solutions  a  loose  plug  of  cotton,  through  which 
the  air  could  freely  circulate,  it  was  found  that  all  suspended  micro- 
organisms could  be  excluded,  and  that  air  passed  through  such  a  filter, 
whether  hot  or  cold,  did  not  cause  fermentation  of  boiled  infusions. 

Similar  results  were  obtained  by  Hoffmann  in  1860,  and  by  Chevreul 
and  Pasteur  in  1861,  without  a  cotton  filter,  by  drawing  out  the  neck 
of  the  flask  to  a  fine  tube  and  turning  it  downward,  leaving  the  mouth 
open.  In  this  case  the  force  of  gravity  prevents  the  suspended  bacteria 
from  ascending,  and  there  is  no  current  of  air  to  carry  them  upward 
through  the  tube  into  the  flask  containing  the  boiled  infusion. 

Tyndall  later  showed  (1876),  by  his  well-known  investigations  upon 
the  floating  matters  of  the  air,  that  in  a  closed  chamber,  in  which  the 
air  is  not  disturbed  by  currents,  all  suspended  particles  settle  to  the 
bottom,  the  superincumbent  air  being  optically  pure,  as  is  proved  by 
passing  a  ray  of  light  through  it.  He  demonstrated  that  the  presence 
of  living  organisms  in  decomposing  fluids  was  always  to  be  explained 
either  by  the  pre-existence  of  similar  living  forms  in  the  infusion  or 
upon  the  walls  of  the  vessel  containing  it,  or  by  the  infusion  having 
been  exposed  to  air  which  was  contaminated  with  organisms. 

These  facts  have  since  been  practically  confirmed  on  a. large  scale 
in  the  preservation  of  food  by  the  process  of  sterilization.  Indeed, 
there  is  scarcely  any  biological  problem  which  has  been  so  satisfactorily 
solved  or  in  which  such  uniform  results  have  been  obtained;  but  all 
through  the  experiments  of  the  earlier  investigators  irregularities  were 
constantly  appearing.  Although  in  the  large  majority  of  cases  it  was 
found  possible  to  keep  boiled  organic  liquids  sterile  in  flasks  to  which 
the  oxygen  of  the  air  had  free  access,  the  question  of  spontaneous 
generation  still  remained  unsettled,  inasmuch  as  occasionally,  even 
under  the  most  careful  precautions,  decomposition  did  occur  in  such 
boiled  liquids. 

This  fact  was  explained  by  Pasteur  in  1860  by  experiments  showing 


HISTORICAL  SKETCH  21 

that  the  temperature  of  boiling  water  was  not  sufficient  to  destroy  all 
living  organisms,  and  that,  especially  in  alkaline  liquids,  a  higher 
temperature  was  required  to  ensure  sterilization.  He  showed  that  at 
a  temperature  of  110°  to  112°  ('.,  however,  which  he  obtained  by  boiling 
under  a  pressure  of  one  and  one-halt'  atmospheres,  all  living  organisms 
were  invariably  killed. 

Pasteur  at  a  later  date  (1865)  demonstrated  that  the  organisms  which 
resist  the  boiling  temperature  are,  in  fact,  reproductive  bodies,  which 
are  now  known  as  .vywr.v. 

In  lN7i>  the  development  of  spores  was  carefully  investigated  and 
explained  by  Ferdinand  Colin.  He,  and  a  little  later  Koch,  showed 
that  certain  rod-shaped  organisms  possess  the  power  of  passing  into 
a  resting  or  spore  stage  under  peculiar  conditions  of  growth,  and  when 
in  this  stage  they  are  much  less  susceptible  to  the  injurious  action  of 
higher  temperatures  than  when  in  their  normal  vegetative  condition. 

With  this  discovery  the  controversy  of  spontaneous  generation,  in  so 
far  as  it  related  to  identified  bacteria,  was  finally  settled.  If  these  micro- 
organisms, some  of  them  being  capable  of  producing  the  more  resistant 
spores,  were  present  in  the  air,  dust,  soil,  water,  etc.,  it  was  easy  enough 
to  explain  the  irregularities  in  the  previous  experiments;  nor  was  it  any 
longer  to  be  doubted  that  these  bacteria,  through  their  products,  were 
the  cause,  not  the  effect,  of  fermentation  and  putrefaction,  and  that 
when  organic  substances  were  completely  sterilized  and  protected  against 
the  entrance  of  living  germs  from  \vithout,  no  development  of  micro- 
organisms occurred  in  them. 

Stimulated  by  the  establishment  of  the  fact,  through  Pasteur's  investi- 
gations, that  fermentation  and  putrefaction  were  due  to  the  action  of 
living  organisms  reproduced  from  similar  pre-existing  forms,  and  that 
each  form  of  fermentation  was  due  to  a  special  micro-organism,  the 
study  of  the  causal  relation  of  micro-organisms  to  disease  was  taken 
up  with  renewed  vigor.  Reference  has  already  been  made  to  the 
opinions  and  hypotheses  of  the  earlier  observers  as  to  the  microbic 
origin  of  infectious  diseases.  The  first  positive  grounds,  however,  for 
this  doctrine,  founded  upon  actual  experiment,  were  the  investigations 
into  the  cause  of  certain  infections  diseases  in  insects  and  plants.  Thus, 
Bassi  in  1837  demonstrated  that  a  fatal  infectious  malady  of  the 
silkworm — muscardine — was  due  to  a  parasitic  micro-organism.  Pasteur 
later  devoted  several  years'  study  to  an  exhaustive  investigation  into 
the  same  subject;  and  in  like  manner  Tulasse  in  1864  and  Kiihne 
in  1855  showed  that  certain  specific  affections  in  grains,  the  potato, 
etc.,  were  due  to  the  invasion  of  parasites. 

Very  soon  after  this  it  was  demonstrated  that  micro-organisms  were 
probably  the  cause  of  certain  infectious  diseases  in  man  and  the  higher 
animals.  Bacteriological  research  has  always  been  of  special  interest 
to  physicians.  Many  of  the  most  distinguished  physicians  of  the  day, 
in  the  earlier  history  of  the  science,  concerned  themselves  in  these 
investigations,  and  the  progress  made  during  the  past  fifteen  or  twenty 
years  has  been  largely  due  to  their  work.  Davaine,  a  famous  French 


22  PRINCIPLES  OF  BACTERIOLOGY 

physician,  has  the  honor  of  having  first  demonstrated  the  causal  relation 
of  a  micro-organism  to  a  specific  infectious  disease  in  man  and  animals. 
The  anthrax  bacillus  was  discovered  in  the  blood  of  animals  dying 
from  this  disease  by  Pollender  in  1849  and  by  Davaine  in  1850;  but 
it  was  not  until  1863  that  the  last-named  observer  demonstrated  by 
inoculation  experiments  that  the  bacillus  was  the  cause  of  anthrax, 
fte,  The  next  discoveries  made  were  those  relating  to  wounds  and  the 
infections  to  which  they  are  liable.  Rindfleisch  in  1866  and  Waldeyer 
and  von  Recklinghausen  in  1871  were  the  first  to  draw  attention  to 
the  minute  organisms  occurring  in  the  pysemic  processes  resulting 
from  infected  wounds,  and  occasionally  following  typhoid  fever.  Further 
investigations  were  made  in  erysipelatous  inflammations  secondary  to 
injury  by  Billroth,  Fehleisen,  and  others,  agreeing  that  in  these  con- 
ditions micro-organisms  could  almost  always  be  detected  in  the  lymph 
channels  of  the  subcutaneous  tissues. 

The  brilliant  results  obtained  by  Lister  in  1863-1870,  in  the  anti- 
septic treatment  of  wounds,  to  prevent  or  inhibit  the  action  of  infective 
organisms,  exerted  a  powerful  influence  on  the  doctrine  of  bacterial 
infection,  causing  it  to  be  recognized  far  and  wide  and  gradually  lessen- 
ing the  number  of  its  opponents.  Lister's  methods  Avere  suggested  to 
him  by  Pasteur's  investigations  on  putrefaction. 

In  1877  Weigert  and  Ehrlich  recommended  the  use  of  the  aniline 
dyes  as  staining  agents  in  the  microscopic  examination  of  micro- 
organisms in  cover-glass  preparations. 

In  the  year  1880  Pasteur  published  his  discovery  of  the  bacillus  of 
fowl  cholera  and  his  investigations  upon  the  attenuation  of  the  virus 
of  anthrax  and  of  fowl  cholera,  and  upon  protective  inoculation  against 
these  diseases.  Laveran  in  the  same  year  announced  the  discovery 
of  parasitic  bodies  in  the  blood  of  persons  sick  with  malarial  fever, 
and  thus  started  investigations  upon  the  unicellular  animal  parasites. 

In  1881  Koch  made  his  fundamental  researches  upon  pathogenic 
bacteria.  He  introduced  solid  culture  media  and  the  "plate  method" 
for  obtaining  pure  cultures,  and  showed  how  different  organisms  could 
be  isolated,  cultivated  independently,  and  by  inoculation  of  pure  cultures 
into  susceptible  animals  made,  in  many  cases,  to  reproduce  the  specific 
disease  of  which  they  were  the  cause.  To  him  more  than  any  other 
are  due  the  methods  which  have  enabled  us  to  prove  absolutely  in  a 
broad  sense  the  permanence  of  bacterial  varieties.  It  was  in  the  course 
of  this  work  that  the  Abbe  system  of  substage  condensing  apparatus 
was  first  used  in  bacteriology. 

In  1882  Pasteur  published  his  first  communication  upon  rabies. 
A  little  later  came  the  investigations  of  Loeffler  and  Roux  upon  the 
diphtheria  bacillus  and  its  toxins,  and  that  of  Kitasato  upon  tetanus. 
These  researches  paved  the  way  for  Behring's  discovery  of  diphtheria 
antitoxin,  which  in  its  turn  stimulated  investigation  upon  the  whole 
subject  of  immunity. 


CHAPTER   II. 

GENERAL  CHARACTERISTICS  OF  BACTERIA— RELATION  TO  OTHER 
MICRO-ORGANISMS—MORPHOLOGY  AND  STRUCTURE. 

BACTERIA  comprise  the  most  important  of  the  groups  of  micro- 
organisms which  have  in  common  the  ability  to  invade  the  living  tissues 
of  animals  and  plants,  and  so  become  involved  in  the  production  of 
disease.  The  micro-organisms  of  some  of  the  groups  are  undoubtedly 
animal  structures,  while  others  are  clearly  minute  plants.  Bacteria 
are  such  primitive  forms  that  their  differentiation  is  not  marked,  being 
related  to  both  plants  and  animals,  but  their  resemblance  to  plants 
seems  to  be  so  much  closer  that  they  are  assigned  to  the  vegetable 
kingdom.  The  bacteria  are  able  to  obtain  their  nourishment  from 
much  simpler  chemical  substances  than  the  animal  cells,  yet  they 
cannot  use  some  of  the  substances  which  are  assimilable  by  the  green 
plants.  Structurally  and  morphologically  they  are  apparently  extremely 
simple,  although  biologically  they  are  very  variable.  Some  bacteria 
are  endowed  with  motility,  others  lack  it.  The  majority  are  reproduced 
by  transverse  division,  and  in  some  respects  they  resemble  the  fungi; 
hence  called  by  Xaegeli  "  fission  fungi,  or  schizomycetes."  They  are 
also  somewhat  allied  to  the  lower  alga?,  especially  in  their  ability  to 
use  simpler  inorganic  substances  to  build  up  higher  compounds,  but 
differ  from  them  in  not  having  chlorophyll.  A  few  varieties  of  unicel- 
lular organisms  resemble  bacteria  in  all  their  known  characteristics, 
except  that  they  possess  chlorophyll  or  substances  similar  to  it.  Others, 
still,  which  have  no  chlorophyll,  are  able,  in  the  absence  of  light,  to 
build  up  organic  substances  synthetically.  The  motile  bacteria  are 
closely  related  to  the  protozoa,  some  of  which  also  invade  animal 
tissues.  The  latter  belong  mostly  to  the  animal  kingdom  and  have 
a  very  wide  distribution. 

The  bacteria  are,  therefore,  a  great  class  of  micro-organisms  which 
have  relation  on  one  or  more  sides  to  other  classes.  There  are  wonderful 
differences  in  the  conditions  of  life  and  nutrition,  which  suit  the  different 
varieties.  We  meet  with  bacterial  life  between  0°  and  75°  C.  Some 
live  only  in  the  tissues  of  men,  others  in  animals,  and  by  far  the  greater 
number  in  dead  organic  matter.  For  some  free  oxygen  is  necessary  to 
life,  for  others  it  is  a  poison. 

Bacteria  may  be  defined  as  extremely  minute,  unicellular  vegetable 
micro-organisms,  which  reproduce  themselves  with  exceeding  rapidity, 
usually  by  transverse  division,  and  nourish  themselves  without  the  aid 
of  chlorophyll.  They  have  great  powers  of  adapting  themselves  to 


24  PRINCIPLES  OF  BACTERIOLOGY 

varied  conditions.  They  have  no  nucleus,  strictly  speaking,  but  con- 
tain a  substance  which  resembles  nuclear  material. 

Those  bacteria  which  depend  entirely  upon  a  living  host  for  their 
existence  are  known  as  strict  parasites;  those  which  can  lead  a  sapro- 
phytic  existence,  but  which  usually  thrive  only  within  the  body  of  a 
living  animal,  are  called  facultative  parasites.  The  strict  saprophytes, 
which  represent  the  large  majority  of  all  bacteria,  while  they  destroy 
refuse  are  not  only  harmless  to  living  organisms,  but  perform  many 
exceedingly  important  functions  in  nature,  such  as  the  destruction  of 
dead  organic  matter  and  its  preparation  for  plant  food  through  decom- 
position, putrefaction,  and  fermentation.  The  parasites,  on  the  contrary, 
are  harmful  invaders  of  the  body  tissues,  exciting  by  their  growth  and 
products  many  forms  of  inflammation  and  disease. 

Relationship  of  Bacteria  to  Other  Micro-organisms. — Bacteria  are 
allied  to  moulds  on  the  one  hand,  and  to  yeasts  on  the  other.  They 
have  no  differentiation  into  root,  stem,  and  leaf.  They  resemble  fungi, 
in  that  no  sexual  reproduction  occurs,  and  that  their  mode  of  multipli- 
cation is  usually  by  simple  division.  From  such  facts  as  these  we  may 
show  their  relations  and  build  up  a  classification  as  follows : 

Thallophyta  (lower  plants  with  no  distinction  between  root  and  stem). 

I 


Forms  with  chlorophyll  Forms  without  chlorophyll, 

(algae,  etc.). 


Multicellular ;  spores  in  differentiated  Unicellular ;  spores  frequently  absent, 

spore-bearing  organs.    (The  true  spore-bearing  cells  little  or  not  at  all 

fungi  and  moulds.)  differentiated ;  no  sexual  reproduction. 


The  bacteria.  The  yeasts 

(blastomycetes). 

The  bacteria  may  be  subdivided  into  lower  and  higher  forms.  The 
lower  forms  are  the  more  numerous  and  consist  of  minute  unicellular 
masses  of  protoplasm.  The  largest  of  the  forms  met  with  in  animals 
are  9,«,  or  micromillimetres  (a  [Jt  =  ^TTOT  °f  an  mcn)>  long  by  less  than 
\t*.  thick.  The  smallest  known  bacteria  measure  0.5/^  long  by  0.2// 
thick,  while  others  are  invisible  with  any  magnification  which  we  now 
possess. 

The  higher  forms  (see  streptothrix)  show  advance  on  the  lower  along 
two  lines:  (1)  On  the  one  hand  they  consist  of  filaments  made  up  of 
simple  elements,  such  as  occur  in  the  lower  forms.  These  filaments 
may  be  more  or  less  septate,  may  be  provided  with  a  sheath,  and  may 
show  branching,  either  true  or  false.  The  minute  structure  of  the 
elements  comprising  these  filaments  is  analogous  to  that  of  the  lower 
forms.  Their  size,  however,  is  often  somewhat  greater.  The  lower 
forms  sometimes  occur  in  filaments,  but  here  every  member  of  the 
filament  is  independent,  while  in  the  higher  forms  there  seems  to  be  a 
certain  interdependence,  among  the  individual  elements.  For  instance, 


GENERAL  CHARACTERISTICS  OF  BACTERIA 


25 


growth  may  occur  only  at  one  end  of  a  filament,  the  other  forming 
an  attachment  to  some  fixed  object.  (2)  The  higher  forms,  moreover, 
present  this  further  development  that  in  certain  cases  some  of  the 
elements  may  be  set  apart  for  the  reproduction  of  new  individuals. 
The  lower  fungi  have  a  still  more  complicate:!  development  (Fig.  1). 

Morphology.  BASK  FORMS  OF  THE  LOWER  BACTERIA. — The  basic 
forms  of  the  single  bacterial  cells  are  threefold — the  sphere,  the  rod,  and 
the  segment  of  a  spiral.  Although  under  different  conditions  the  type 
form  of  any  one  species  may  vary  considerably,  yet  these  three  main 
divisions  under  similar  conditions  are  permanent;  and,  so  far  as  we 
know,  it  is  never  possible  by  any  means  to  bring  about  changes  in  the 

EJG.  1 


1,  branched  filament  carrying  spores;  2,  cross-section  of  spore  highly  magnified;  3  and  4,  spore 
building  ;  5,  developing  and  bursting  spores ;  6and7,  branching  ;  8,  sprouting  spores.    (After  Tavel.) 


organisms  that  will  result  in  the  conversion  of  the  morphology  of  the 
members  of  one  group  into  that  of  another — that  is,  micrococci  always, 
under  suitable  conditions,  produce  micrococci,  bacilli  produce  bacilli, 
and  spirilla  produce  spirilla. 

The  form  of  the  bacterial  cells  at  their  stage  of  complete  development 
must  be  distinguished  from  that  which  they  possess  just  after  or  just 
before  they  have  divided.  As  the  spherical  cell  develops  preparatory 
to  its  division  into  two  cells  it  becomes  elongated  and  appears  as  a 
short  oval  rod;  at  the  moment  of  its  division,  on  the  contrary,  the  trans- 
verse diameter  of  each  of  its  two  halves  is  greater  than  their  long 
diameter.  A  short  rod  becomes  in  the  same  way,  at  the  moment  of 
its  division,  two  cells,  the  long  diameter  of  each  of  which  may  be 


26  PRINCIPLES  OF  BACTERIOLOGY 

even  a  trifle  less  than  its  short  diameter,  and  thus  they  appear  on  super- 
ficial examination  as  spheres. 

As  bacteria  multiply  the  cells  produced  from  the  parent  cell  have 
a  greater  or  less  tendency  to  remain  attached.  This  is  on  account  of 
the  slimy  envelope  which  all  bacteria  have  more  or  less  developed.  In 
some  varieties  this  tendency  is  extremely  slight,  in  others  it  is  marked. 
This  union  may  appear  simply  as  an  aggregation  of  separate  bacteria 
or  so  close  that  the  group  appears  as  a  single  cell.  According  to  the 
method  of  the  cell  division  and  the  tenacity  with  which  the  cells  hold 
together,  we  get  different  groupings  of  bacteria,  which  aid  us  in  their 
differentiation  and  identification.  Thus,  whether  the  bacterial  cell 
divides  in  one,  two,  or  three  planes,  we  get  forms  built  in  one,  two,  or 
three  dimensions.  If  we  group  bacteria  according  to  the  characteristic 
form  of  the  cells,  and  then  subdivide  them  according  to  the  manner  of 
their  division  in  reproduction  and  the  tenacity  with  which  the  newly 
developed  cells  cling  to  one  another,  we  will  have  the  following  varieties : 


FIG.  2 


::::    .» 

::::   * 


r/ 


« 


^nn 


Varieties  of  spherical  forms  :   a,  tendency  to  lancet-shape  ;  b,  tendency  to  coffee-beau  shape  ;  c,  in 
packets ;  d,  in  tetrads ;  e,  in  chains ;  /,  in  irregular  masses.    (After  Fliigge.) 

1.  SPHERICAL  FORM,  OR  Coccus  (Fig.  2).— The  size  varies  from 
about  0.3/J.  as  minimum  diameter  to  3/*  as  maximum.  The  single 
elements  are  at  the  moment  of  their  complete  development,  so  far  as 
we  can  determine,  absolutely  spherical;  but  when  seen  in  the  process 
of  multiplication  through  division  the  form  is  seldom  that  of  a  true 
sphere.  Here  we  have  elongated  or  lancet-shaped  forms,  as  frequently 
seen  in  the  diplococcus  of  pneumonia,  or  the  opposite,  as  in  the  diplo- 
coccus  of  gonorrhoea,  where  the  cocci  appear  to  be  flattened  against 
one  another.  Those  cells  which  divide  in  one  direction  only  and  remain 
attached  are  found  in  pairs  (diplococci)  or  in  shorter  or  longer  chains 
(streptococci).  Those  which  divide  in  two  directions,  the  one  at  right 
angles  to  the  other,  form  bunches  of  four  (tetrads).  Those  which 
divide  in  three  directions  and  cling  together  form  packets  in  cubes 
(sarcinre).  Those  which  apparently  divide  irregularly  in  any  axis 
form  irregularly  shaped,  grape-like  bunches  (staphylococci). 

There  are  a  considerable  number  of  bacteria  which  appear  to  fre- 
quently assume  spherical  forms,  or  at  least  forms  so  like  spheres  that 
they  cannot  be  differentiated  from  them,  and  yet  under  other  conditions 


<;!•:. \ERAL  CHARACTERISTICS  OF  BACTERIA 


27 


they  generate  rod-like  forms.  These  apparently  spherical  bacteria  we  can 
properly  regard  as  short  forms  of  bacilli,  which,  owing  to  the  rapidity  of 
division,  are  for  the  time  being  of  the  same  size  in  both  diameters.  Under 
suitable  conditions,  however,  the  true  rod-shape  is  always  developed. 


FIG.  3 


W*f 


Various  forms  of  bacilli :  a,  bacilli  with  sides  parallel  to  their  Ion?  axis  and  with  ends  perpen- 
dicular ;  6,  bacilli  with  sides  swollen  or  narrowed,  causing  irregular  forms.     (Alter  FlUgge.) 

2.  ROD  FORM,  OR  BACILLUS. — The  type  of  this  group  is  the  cylinder. 
The  length  of  the  fully  developed  cell  is  always  longer  than  its  breadth. 
The  size  of  the  cells  of  different  varieties  varies  enormously,  from  a  length 
of  30u  and  a  breadth  of  4u  to  a  length  of  0.2,«  and  a  breadth  of  O.I/*. 
The  largest  bacilli  met  with  in  disease  do  not,  however,  average  over 
3«.  In  describing  their  forms  bacilli  are  roughly  classed  as  slender 


FIG.  4 


FIG.  5 


Medium  bacilli,  single  and  in  pairs. 


Small  bacilli,  mostly  in  pairs. 


when  the  ratio  of  the  long  to  the  transverse  diameter  is  from  1 : 4  to 
1 : 10,  and  as  thick  when  the  proportions  of  the  long  to  the  short  diameter 
is  approximately  1 : 2. 

The  characteristic  form  of  the  bacillus  is  one  with  a  straight  axis, 
uniform  thickness  throughout,  and  flat  ends  (Fig.  3  a,  and  Fig.  6);  but 


'28 


PRINCIPLES  OF  BACTERIOLOGY 


there  are  many  exceptions  to  this  typical  form.  Thus  frequently  the 
motile  bacteria  have  rounded  ends  (Fig.  3) ;  many  of  the  more  slender 
forms  have  the  long  axis  bent;  some  few  species,  such  as  the  diphtheria 
bacilli  (Fig.  4),  invariably  produce  many  cells  whose  thickness  is  very 
unequal  at  different  portions.  Spore  formation  also  causes  an  irregu- 
larity of  the  cell  outline  (Figs.  7,  17  and  18). 


FIG.  7 


Large  bacilli  in  chains. 


Spores    in    centre    of    bacilli. 
(From  Kolle  and  Wasserman.) 


The  bacilli  except  when  they  develop  from  spoies  or  granules  divide 
only  in  the  plane  perpendicular  to  their  long  axis.  A  classification, 
therefore,  of  bacilli  according  to  their  manner  of  grouping  is  much 
simpler  than  in  the  case  of  the  cocci.  We  may  thus  have  bacilli  as 
isolated  cells,  as  pairs,  or  as  longer  or  shorter  chains. 

3.  SPIRAL  FORM,  OR  SPIRILLUM. — The  members  of  the  third  mor- 
phological group  are  spiral  in  shape,  or  rather  segments  of  a  spiral. 
Here,  too,  we  have  large  and  small,  slender  and  thick  spirals.  The 


FIG.  8 


FIG. 


Medium-sized  spirilla. 


twisting  of  the  long  axis,  which  here  lies  in  two  planes,  is  the  chief 
characteristic  of  this  group  of  bacteria.  Under  normal  conditions  the 
twisting  is  equal  throughout  the  entire  length  of  the  cell.  The  spirilla, 
like  the  bacilli,  divide  only  in  one  direction.  A  single  cell,  a  pair,  or  the 
union  of  two  or  more  elements  may  thus  present  the  appearance  of  a 
short  segment  of  a  spiral  or  a  comma-shaped  form,  an  S-shaped  form, 
or  a  complete  spiral  or  corkscrew-like  form  (Figs.  8,  9,  10  and  11). 


(;i-:\ERAL  CHARACTERISTICS  OF  BACTERIA  29 

The  determination  of  morphological  characters  for  the  description 
of  bacteria  should  always  be  made  from  fully  developed  cultures;  those 
which  are  too  young  may  present,  as  already  noted,  immature  forms, 
due  to  rapid  multiplication,  while  in  old  cultures  altered  or  degenerated 
forms  may  be  observed. 

A.Vlien  growth  is  obtained  upon  different  media,  variations,  especially 
in  size,  may  sometimes  be  observed.  These  differences  should  always 

FIG.  10  FIG.  11 

„••?  /"'"  •• 

- 


be  described,  together  with  a  note  of  the  media  upon  which  they  were 
developed  and  a  statement  as  to  whether  such  variation  is  a  marked 
feature  of  the  species  under  consideration. 

The  conditions  of  temperature  and  of  nutrition  which  favor  growth 
are  very  various  for  different  species,  so  that  no  fixed  temperature, 
medium,  or  age  of  growth  can  be  determined  upon  as  applicafjle  to  all 

FIG.  12  FIG.  13 


-d 

{——•c 


Structure  of  bacterial  cells.  Plasmolysis :  a,  spirillum  undula  ;  6,  bacillus  solKusii ;  c,  vibrio 

(After  BUtschli.)  cholene.    (After  A.  Fischer.) 

species.  Morphological  descriptions  should  always  be  accompanied 
by  a  definite  statement  of  the  age  of  the  growth,  the  medium  from 
which  it  was  obtained,  and  the  temperature  at  which  it  was  developed. 
Structure  of  Bacterial  Cells. — When  examined  in  water  under  the 
microscope  bacteria  appear  merely  as  colorless  refractile  bodies  writh 
or  without  spores.  It  is  only  through  using  special  stains  that  wre  are 
able  to  see  more  of  their  structure  (Fig.  12).  They  are  thus  found  to 


30  PRINCIPLES  OF  BACTERIOLOGY 

have  a  rather  indefinite  cell  membrane  surrounding  the  central  proto- 
plasm. This,  according  to  Zettnow,  contains  a  nucleus,  or  at  least 
the  equivalent  of  the  nucleus  of  the  higher  micro-organisms,  lying 
within  a  network  of  protoplasma.  The  nuclear  substance  takes  the 
chromatin  stains  and  is  often  so  abundant  that  the  material  holding  it  is 
covered  up.  The  plasma  substance  divides  into  the  entoplasma  and  the 
ectoplasma.  The  first  is  more  or  less  intimately  connected  with  the 
nuclear  substance,  and  is  especially  collected  at  the  ends  of  the  long 
bacteria.  This  stains  blue  by  the  Romanowski  method.  The  ecto- 
plasma, or  cell  membrane,  is  a  much  more  concentrated  substance  than 
the  entoplasma,  and  remains  unstained  ordinarily,  but  by  special  stains 
appears  as  an  external  shell  or  flagella.  The  membrane  surrounding 
some  bacteria  is  very  slightly  developed ;  in  others,  as  in  tubercle  bacilli, 
it  is  well  developed.  It  is  never  a  cellulose  envelope  like  the  higher 
plant  cells,  but  by  means  of  its  chemical  composition  is  differentiated 
from  the  inner  plasma,  as  shown  in  plasmolysis.  Thus,  when  bacteria 
are  placed  in  1  per  cent,  chloride  of  sodium  the  central  body  contracts 
and  separates  itself  in  places  from  the  capsule,  as  shown  in  Fig  13. 

THE  CAPSULF. — The  capsule  consists  of  an  inner  tougher  portion 
immediately  surrounding  the  central  body  and  which  gradually  passes 
into  a  thinner  and  more  watery  outer  portion  which  is  uncolored  by 
ordinary  staining  method. 

This  is  indicated  by  the  greater  apparent  diameter  of  bacteria  when 
stained  with  certain  dyes  beyond  what  they  usually  show.  Certain 

bacteria,    however,   commonly   known    as 
Fl°- 14  capsule  bacteria,  as  shown  in  Fig.  14,  have 

the  outer  layers  of  the  membrane  so  much 
thickened  that  the  bacteria  seem  to  be 
surrounded  by  a  broad  gelatinous  envelope 
or  capsule,  which  is  distinguished  by  a 
diminished  power  of  staining  with  the  or- 
dinary aniline  dyes.  The  foim  of  the  cap- 
sule varies  with  different  species  (Fig.  12). 
The  demonstration  of  this  capsule  is  often 
of  help  in  differentiating  between  certain 
bacteria — e.  g.,  some  forms  of  the  strep- 
capsule  tococcus  and  pneumococcus.  A  peculi- 
formation.  arity  of  the  capsule  bacteria  is  that,  except 

very  rarely,  they  exhibit  this  envelope  only 
when  grown  in  the  animal  body  or  in  special  culture  media,  such  as 
milk,  blood  serum,  bronchial  mucus,  etc.;  grown  on  nutrient  gelatin 
agar  or  potato  the  capsule  is  only  visible  under  very  exceptional  con- 
ditions, and  then  not  distinctly. 

ORGANS  OF  MOTILITY. — The  outer  surface  of  bacteria,  when  occur- 
ring in  the  form  of  spheres,  is  almost  always  smooth  and  devoid 
of  appendages ;  but  the  rods  and  spirals  are  frequently  provided  with 
fine,  hair-like  appendages,  or  flagella,  which  are  their  organs  of  motility 
(Figs.  15  and  16).  These  flagella,  either  singly  or  in  numbers,  are 


GENERAL  CHARACTERISTICS  OF  BACTERIA  31 

sometimes  distributed  over  the  entire  body  of  the  cell,  or  they  may 
form  a  tuft  at  one  end  of  the  rod,  or  only  one  polar  flagellum  is  found. 
The  polar  flagella  appear  on  the  bacteria  shortly  before  division. 
The  flagella  are  believed  to  be  formed  of  the  ectoplasma,  and  proba- 
bly have  the  property  of  protrusion  and  retraction.  So  far  as  we 
know,  the  flagella  are  the  only  means  of  locomotion  possessed  by  the 
bacteria.  They  are  not  readily  stained,  special  staining  agents  being 
required  for  this  purpose.  The  envelope  of  the  bacteria,  which  usually 
remains  nmstained  with  the  ordinary  dyes,  then  becomes  colored  and 
more  distinctly  visible  than  is  commonly  the  case.  Occasionally, 
however,  some  portion  of  the  envelope  remains  unstained,  when  the 
flagella  present  the  appearance  of  being  detached  from  the  bodyfof 
the  bacteria  by  a  narrow  zone.  In  cultures  of  richly  flagellated  bacteria 
peculiar  pleated  masses  sometimes  are  observed,  consisting  of  flagella 
which  have  been  detached  and  then  matted  together.  Bacteria  may 
lose  their  power  of  producing  flagella  for  a  series  of  generations.  Whether 
their^  power  be  permanently  lost  or  not  we  do  not  know. 

FIG.  15  Fro   ifi 

Jfc;  . 


rf? 
>%•      v 


Bacilli  showing  one  polar  flagellum.  Bacilli  showing  multiple  flagella. 

REPRODUCTION  AMONG  THE  LOWER  BACTERIA. — When  a  bacterial 
cell  is  placed  in  favorable  surroundings  for  development  it  multiplies, 
as  a  rule,  by  simple  division.  When  such  development  is  in  progress  a 
single  cell  will  be  seen  to  elongate,  in  the  case  of  spherical  bacteria  only 
slightly,  and  in  the  rod-shaped  organisms  considerably  in  one  direction. 
Over  the  centre  of  the  long  axis  thus  formed  will  appear  a  slight  inden- 
tation in  the  outer  envelope  of  the  cell;  this  indentation  increases  in 
extent  until  there  exists  eventually  two  individuals.  As  a  rule,  the  cells 
separate  from  one  another  soon  after  division,  but  occasionally  they 
remain  together  for  a  time,  forming  pairs  and  chains.  Under  certain 
conditions  of  nutrition  long  threads  or  filaments  are  formed,  which, 
however,  when  put  in  contact  with  new  food,  break  up  into  fragments. 
At  times,  when  the  culture  media  are  exhausted  or  nearly  so,  the  bacilli 
and  spirilla  will  be  found  to  «go  on  dividing,  with  little  or  no  increase 


32  PRINCIPLES  OF  BACTERIOLOGY 

in  length,  and  thus  coccus-like  forms  result;  but  when  these  are  given 
fresh  food  under  suitable  conditions  they  elongate  and  reproduce 
the  usual  shaped  organisms.  According  to  recent  investigations  on 
the  subject  of  cell  reproduction,  the  division  of  the  cell  starts  from  the 
protoplasmic  layer,  the  central  space  being  passively  destroyed,  and  the 
outer  envelope  is  only  secondarily  concerned  in  the  process.  This  would 
indicate  that  the  central  space  is  not  a  true  nucleus,  otherwise  the 
division  of  the  nucleus  should  precede  the  cell  division.  The  complete 
process  of  cell  reproduction  in  most  varieties  occupies,  under  favorable 
conditions,  about  twenty  to  thirty  minutes. 

But  although  elongation  in  the  greater  diameter  and  transverse 
division  is  the  rule  for  the  majority  of  bacteria,  there  are  certain  groups, 
as  the  sarcinse,  for  example,  which  divide  more  or  less  regularly  in 
three  directions.  Instead  of  becoming  separated  from  each  other  as 
single  cells,  the  tendency  then  is  for  the  segmentation  to  be  incomplete, 
the  cells  remaining  together  in  masses.  The  indentations  upon  these 
masses  or  cubes,  which  indicate  the  point  of  incomplete  fission,  give  to 
these  bundles  of  cells  the  appearance  commonly  ascribed  to  them— 
that  of  a  bale  of  rags.  As  already  said,  division  in  two  opposite  direc- 
tions results  in  the  formation  of  a  group  of  forms  as  tetrads.  Division 
irregularly  in  all  directions  results  in  the  production  of  clusters.  The 
rod-shaped  bacteria  never  divide  longitudinally. 

REPRODUCTION  AMONG  THE  HIGHER  BACTERIA. — Most  of  the  higher 
bacteria  consist  of  thread-like  structures  more  or  less  septate  and  often 
surrounded  by  a  sheath.  The  organism  is  frequently  attached  at  one 
end  to  some  object  or  to  another  individual.  It  grows  to  a  certain  length, 
and  then  at  the  free  end  certain  cells  called  gonidia  are  cast  off,  from 
which  new  individuals  are  formed.  The  gonidia  do  not  possess  any 
special  powers  of  resistance. 

Spore  formation  must  be  distinguished  from  vegetative  reproduction. 
This  is  the  process  by  which  the  organisms  are  enabled  to  enter  a  stage 
in  which  they  resist  deleterious  influences  to  a  much  higher  degree  than 
is  possible  for  them  in  the  growing  or  vegetative  condition.  It  is  true 
that  in  all  cultures  a  certain  proportion  of  the  bacteria  are  more  resistant 
than  the  average.  No  difference  in  protoplasm,  however,  has  been 
noted  in  them.  They  are  probably  the  youngest  and  most  hardy  or 
perhaps  involution  forms.  The  difference  between  these  and  the  less 
resistant  forms  is  not  great.  Some  have  believed  that  this  resistance  was 
due  to  certain  bodies  called  arthrospores,  or  jointed  spores,  developed 
not  within  the  cell,  but  as  a  sprout-like  separation  of  one  of  its  extremi- 
ties. Recent  researches  into  the  formation  of  arthrospores  have 
resulted  in  questioning  their  existence.  There  is,  therefore,  in  the  lower 
bacteria,  only  one  kind  of  spores  requiring  special  notice — viz.,  endo- 
spores.  These  are  strongly  refractile  and  glistening  in  appearance, 
oval  or  round  in  shape,  and  composed  of  concentrated  protoplasm 
developed  within  the  cell  (Figs.  17  and  18).  They  are  characterized 
by  the  power  of  resisting  the  injurious  influences  of  heat,  desiccation, 
and  chemical  disinfectants. 


GENERAL  CHARACTERISTICS  OF  BACTERIA 


33 


The  production  of  endospores  in  the  different  species  of  bacteria, 
though  not  identical,  is  very  similar.  To  observe  the  formation  of 
spores  in  any  species  it  is  best  to  employ  a  streak  culture  on  nutrient 
agar  or  a  potato  culture,  which  should  be  kept  at  the  temperature  nearest 
the  optimum  of  the  organism  to  be  examined.  At  the  end  of  twelve, 
eighteen,  twenty-four,  thirty,  thirty-six  hours,  etc.,  specimen's  of  the 
culture  are  observed  first  unstained  in  a  hanging  drop  or  agar  block, 
and  then,  if  round  or  oval,  highly  refractile  bodies  are  seen,  they  should 
be  stained  for  spores.  A  bacillus,  as  a  rule,  produces  but  one  spore,  and 
more  than  two  have  never  been  observed. 

According  to  Fischer  motile  bacteria  always  come  to  a  state  of  rest 
or  immobility  previous  to  spore  formation.  Several  species  first  become 
elongated.  The  anthrax  bacillus  does  this,  and  a  description  of  the 
method  of  its  production  of  spores  may  serve  as  an  illustration  of  the 
process.  In  the  beginning  the  protoplasm  of  the  elongated  filaments 
is  homogeneous,  but  after  a  time  it  becomes  turbid  and  finely  granular. 


FJG.  17 


FIG.  18 


Unstained  spores  in  slightly  distended 
bacilli.  (The  spores  are  the  light  oval 
spaces  in  the  heavily  stained  bacilli.) 


Unstained  spores  in  distended  ends  of 
bacilli. 


These  fine  granules  are  then  replaced  by  a  smaller  number  of  coarser 
granules,  which  are  finally  amalgamated  into  a  spherical  or  oval  refrac- 
tile body.  This  is  the  spore.  As  soon  as  the  process  is  completed  there 
appears  between  two  spores  a  delicate  partition  wall.  For  a  time  the 
spores  are  retained  in  a  linear  position  by  the  cell  membrane  of  the 
bacillus,  but  this  is  later  dissolved  or  broken  up  and  the  spores  are  set 
free.  Not  all  the  cells  that  make  the  effort  to  form  spores,  as  shown  by 
the  spherical  bodies  contained  in  them,  bring  these  to  maturity;  indeed, 
many  varieties,  under  certain  cultural  conditions,  lose  their  property  of 
forming  spores.  The  following  are  the  most  important  spore  types: 
(a)  the  spore  lying  in  the  interior  of  single,  short,  undistended  cells; 
/'  the  spores  lying  in  the  interior  of  a  chain  of  undistended  cells 
Fig.  7)  ;  (c)  the  spore  lying  at  the  extremity  of  a  cell  much  enlarged 
at  that  end  —  the  so-called  "head  spore  "  (Fig.  18);  and  (d)  the  spore 
lying  in  the  interior  of  a  cell  very  much  enlarged  in  its  central  portion, 
giving  it  a  spindle  shape. 

3 


34 


PRINCIPLES  OF  BACTERIOLOGY 


The  germination  of  spores  (Fig.  19)  takes  place  as  follows:  By  the 
absorption  of  water  they  become  swollen  and  pale  in  color,  losing  their 
shining,  refractile  appearance.  Later,  a  little  protuberance  is  seen  upon 
one  side  or  at  one  extremity  of  the  spore,  and  this  rapidly  grows  out  to 


FIG.  19 


FIG.  20 


123 

Showing  methods  of  spore  germination  :  .1,  polar  germination  of  B.  butyricus  (after  Prazmowski); 
B,  equatorial  germination  of  B.  subtilis  (after  Prazmowski);  C-D,  equatorial  germination  of  B. 
tumescens  and  of  Bact.  carotorum  (after  A.  Koch);  E-F,  polar  germination  of  Bact.  sessile  (after 
L.  Klein);  H,  germination  by  absorption  of  B.  anthracis  (after  De  Bary);  G,  endogermination  in 
spirillum  endoparagogicum  (after  Sorokin);  1-K,  spore  formation  in  Bact.  anthracis  (after  Migula). 

form  a  rod  which  consists  of  soft-growing  protoplasm  enveloped  in  a 
membrane,  which  is  formed  of  the  endosporium  or  inner  layer  of  the 

cellular  envelope  of  the  spore.  The  outer 
envelope,  or  exosporium,  is  cast  off  and 
may  be  seen  in  the  vicinity  of  the  newly 
formed  rod.  Sometimes  the  vegetative  cell 
emerges  from  one  extremity  of  the  oval 
spore,  and  in  other  species  the  exosporium 
is  ruptured  and  the  bacillus  emerges  from 
the  side. 

INVOLUTION  FORMS. — In  old  cultures  of 
bacteria,  in  old  abscesses,  and  in  other 
places  where  the  deleterious  substances 
have  developed  and  the  foodstuffs  have 
been  largely  used,  the  rapidity  of  division 
of  the  bacteria  is  lessened  and  there  are 
frequently  found  very  irregular  or  distorted 
forms,  due  to  the  abnormal  development 
and  division  of  the  bacterial  cells  under  the  unfavorable  conditions 
present.  These  are  spoken  of  as  involution  or  degenerated  forms.  If 
these  deformed  cells  are  placed  under  suitable  conditions  they  pro- 
duce again  normally  fashioned  organisms.  Cocci  may  produce  irregular 
rods  and  bacilli  threads.  In  old  abscesses  the  involution  forms  may 
look  entirely  different  from  the  well-developed  cells. 

METACHROMATIC  GRANULES. — These  appear  in  unstained  bacteria 
as  light-refracting,  in  stained  preparations  as  deeply  stained,  granules. 


Involution  forms  from  bacilli. 
(From  Fliigge.) 


CHARACTERISTICS  OF  BACTERIA  35 

They  have  a  great  affinity  for  dyes,  and  so  stain  readily  and  give  up 
the  stain  with  some  difficulty.  In  certain  bacteria,  such  as  the  diphtheria 
bacilli,  they  are  especially  well  marked.  Here  they  have  diagnostic 
value.  Besides  the  metachromatic  granules  there  are  others  which  take 
up  stains  with  difficulty. 


CHAPTEE    III. 

THE  CLASSIFICATION  OF  BACTERIA— PERMANENCE   OF  SPECIES 
—CHEMICAL  COMPOSITION  AND  NUTRITION. 

Genera  and  Species. — Bacteria  have  been  classified  in  many  different 
ways  by  different  observers.  As  a  rule  the  genera  are  based  upon  mor- 
phological characters  and  the  species  upon  biochemical,  physiological, 
or  pathogenic  properties.  While  the  form,  size,  and  method  of  division 
are  the  most  permanent  characteristics  of  bacteria,  and  so  are  naturally 
utilized  for  classification,  nevertheless,  in  this  basis  of  division  there  are 
decided  difficulties.  Thus,  while  the  form  and  size  of  bacteria  are  fairly 
constant  under  the  same  conditions,  we  have  already  noticed  that  they 
may  be  quite  different  under  diverse  conditions.  Another  serious  draw- 
back for  our  purposes  is  that  these  morphological  characteristics  give 
no  indication  whatever  of  the  relations  of  the  bacteria  to  disease  and 
fermentation — the  very  characteristics  for  which  as  physicians  we  study 
them.  Other  properties  of  bacteria  which  are  fairly  constant  under 
uniform  conditions  are  those  of  spore  and  capsule  formation,  motility, 
reaction  to  staining  reagents,  relation  to  temperature,  to  oxygen,  and 
other  food  material,  and,  finally,  their  relation  to  fermentation  and 
disease. 

Taking  any  one  of  these  properties  of  bacteria  as  a  basis,  we  can 
classify  them;  but  even  here  there  will  be  groups  which  under  certain 
conditions  would  be  placed  in  one  class  and  under  others  in  another. 

Thus,  the  power  to  produce  spores  may  be  totally  lost  or  held  in 
abeyance  for  a  time. 

The  relations  to  oxygen  may  be  gradually  altered,  so  that  an  anaerobic 
species  grows  in  the  presence  of  oxygen.  Parasitic  bacteria  may  be  so 
cultivated  as  to  become  saprophytic  varieties,  and  those  which  have 
no  power  to  grow  in  the  living  body  may  acquire  pathogenic  proper- 
ties. 

The  possibility  of  making  any  thoroughly  satisfactory  classification 
is  rendered  still  more  difficult  by  the  fact  that  many  necessarily  imper- 
fect attempts  have  already  been  made,  so  tha,t  there  is  a  great  deal 
of  confusion,  which  is  steadily  increased  as  new  varieties  are  found 
or  old  ones  reinvestigated  and  classified  differently  in  the  different 
systems. 

As  one  of  the  more  successful  attempts  to  classify  bacteria,  the  system 
devised  by  Migula  is  here  given,  simply  as  an  example.  The  morphology 
of  bacteria  is  used  as  the  basis  of  the  division: 


THE  CLASSIFICATION  OF  BACTERIA  37 

I    \MILIES. 

I.   Cells  globose  in  a  free  state,  not  elongating  in  any 

direction  before  division  into  1,  2,  or  3  planes      .     1.  Coccacese. 
II.  Cells  cylindrical,  longer  or  shorter,  and  only  divid- 
ing in  one  plane,  and  elongating  to  about  twice  the 
normal  length  before  the  division, 
a.  Cells    straight,  rod-shaped,  without    sheath,  non- 
motile,  or  motile  by  means  of  flagella  .         .         .     2.   Bacteriaceae. 
6.  Cells  crooked,  without  sheath          .        .        .        .3.  Spirillaceaj. 
c.  Cells  enclosed  in  a  sheath 4.  Chlamydobacteriaceae. 

GENERA. 

1.   Coccacece. 
Ceils  without  organs  of  motion. 

a.  Division  in  one  plane 1.  Streptococcus. 

b.  Division  in  two  planes 2.  Micrococcus. 

c.  Division  in  three  planes         .         .         .         .         .3.   Sarcina. 
Cells  with  organs  of  motion. 

a.   Division  in  two  planes 4.   Planococcus. 

6.  Division  in  three  planes 5.  Planosarcina. 

2.  Bacteriacece. 

Cells  without  organs  of  motion       .         .         .         .         .1.  Bacterium. 
Cells  with  organs  of  motion  (flagella). 

a.  Flagella  distributed  over  the  whole  body        .         .2.  Bacillus. 

b.  Flagella  polar 3.  Psehdomonas. 

3.  Spiriltacece. 
Cells  rigid,  not  snake-like  or  flexuous. 

a.  Cells  without  organs  of  motion        .         .         .         .1.  Spirosoma. 
6.  Cells  with  organs  of  motion  (flagella). 

1.  Cells  with  1,  very  rarely  2  to  3  polar  flagella  .     2.  Microspira. 

2.  Cells  with  polar  flagella-tufts           ...     3.  Spirillum. 
Cells  flexuous 4.  Spirochseta. 

4.   ChlamydobacteriacecB  (higher  bacteria). 

Cell  contents  without  granules  of  sulphur. 

a.  Cell  threads  unbranched. 
I.  Cell  division  always  only  in  one  plane      .  .1.  Streptothrix. 

II.  Cell  division  in  three  planes  previous  to  the  forma- 
tion of  gonidia. 

1.  Cells   surrounded   by   a   very   delicate,  scarcely 

visible  sheath  (marine)         .....     2.   Phragmidiothrix. 

2.  Sheath  clearly  visible  (in  fresh  water)    .         .         .3.  Crenothrix. 

6.  Cell  threads  branched      .         .         .         .         .4.  Cladothrix. 
Cell  contents  containing  sulphur  granules      .         .         .5.  Thiothrix. 

The  above  table  makes  changes  in  the  designation  of  some  of  the  most 
common  bacteria,  as  in  the  restoration  of  the  old  title  bacterium  and  the 
assigning  to  it  of  all  non-motile,  rod-shaped  organisms,  thus  altering 
the  name  of  some  of  the  most  common  pathogenic  bacteria  from  bacillus 
to  bacterium.  Other  changes  are  seen  in  the  spirilla.  Any  such  scheme 


38  PRINCIPLES  OF  BACTERIOLOGY 

is  at  times  arbitrary  in  placing  some  varieties  under  one  generic  division 
and  others  closely  allied  in  another.  It  has  also  the  objection,  already 
noted,  that  it  is  only  one  of  several  classifications  already  in  use,  and 
until  an  authoritative  body  agrees  on  some  one  it  seems  unwise  in 
such  a  volume  as  this  to  change  the  usually  employed  names  for 
others  which  are,  perhaps,  intrinsically  better.  Another  important 
reason  for  waiting  is  that  with  the  increase  of  our  knowledge  we  are 
constantly  changing  the  position  of  different  bacteria.  Thus,  such  a 
well-known  germ  as  the  tubercle  bacillus  is  now  found  to  produce, 
under  certain  conditions,  long,  thread-like  branching  forms ;  so  that  it 
ceases  to  be  under  the  classification  of  Migula,  either  a  bacillus  or  bac- 
terium. We  will,  therefore,  simply  use  in  this  book  the  older,  less  scien- 
tific nomenclature,  of  classing  all  rod  forms  as  bacilli  and  all  spiral 
forms  as  spirilla,  and  consider  together,  in  so  far  as  is  practicable,  cer- 
tain groups  of  bacteria  whose  members  are  closely  allied  to  each  other 
in  some  one  or  more  important  directions. 

Permanence  of  Bacterial  Species. — When  we  come  to  study  special 
varieties  or  groups  of  bacteria,  such  as  the  bacilli  which  produce  typhoid 
fever,  diphtheria,  and  tuberculosis,  it  is  of  great  importance  for  us  to 
determine,  if  possible,  to  what  extent  the  peculiar  characteristics  which 
each  of  these  groups  of  bacteria  possess  are  permanent  in  the  generations 
which  develop  from  them. 

We  cannot  believe  that  the  multitude  of  bacterial  varieties  which 
now  exist  have  always  existed.  The  probability  is  very  strong  that 
with  succeeding  generations  and  changing  conditions  new  bacterial 
varieties  have  developed  with  new  characteristics. 

From  time  to  time  the  changing  conditions  under  which  life  progressed 
probably  exposed  certain  animals  to  the  invasion  of  varieties  which 
never  before  had  gained  access  to  them.  If  the  bacteria  found  some 
means  of  transmission  to  other  animals  equally  susceptible,  a  parasitic 
species  became  established  which  at  first,  perhaps,  found  conditions 
only  occasionally  favorable  to  it.  Thus  in  some  such  way  a  multitude 
of  bacterial  groups  arose,  some  of  which  accustomed  themselves  to 
the  conditions  present  in  living  plants,  others  to  those  in  fishes,  others 
to  those  in  birds,  and  others  still  to  those  in  man  and  the  higher 
animals. 

These  are,  however,  theories.  W7hat  has  been  actually  observed  in  the 
few  years  during  which  bacteria  have  been  studied?  In  this  short  time 
the  pathogenic  species  as  observed  in  disease  have  remained  practically 
unaltered.  The  diphtheria  bacilli  are  the  same  to-day  as  when  Loeffler 
discovered  them  in  1884,  and  the  disease  itself  is  evidently  the  same  as 
history  shows  it  to  have  been  before  the  time  of  Christ.  The  same 
permanence  of  disease  type  is  true  for  tuberculosis,  smallpox,  hydro- 
phobia, leprosy,  etc.  Under  practically  unchanged  conditions,  there- 
fore, as  exist  in  the  bodies  of  men,  bacteria  which  have  once  become 
established  as  parasites  continue,  so  long  as  they  remain,  to  retain  their 
peculiar  (specific)  characteristics.  Whether  new  disease  varieties,  such 
as  the  influenza  bacillus,  are  coming  into  existence  from  time  to  time,  is, 


THE  CLASSIFICATION  OF  BACTERIA  39 

of  course,  a  possibility,  hut  not  a  certainty.  The  one  thing  we  can 
probably  safely  assert  is  that  there  is  no  probability  that  any  saprophytir 
variety  now  existing  can,  under  any  possibility,  develop  into  the  now 
recognized  varieties  of  pathogenic  bacteria.  It  is  almost  impossible  to 
conceive  that  any  such  variety  should  develop  parasitic  tendencies ,  under 
exactly  the  same  circumstances  as  those  varieties  which  now  produce 
disease. 

The  fact  that  the  chief  pathogenic  varieties  of  bacteria,  which  excite 
disease  in  man,  seem  to  have  retained  for  centuries  their  characteristics, 
in  no  way  proves  that  when  placed  under  different  conditions  they  would 
remain  stable.  As  already  stated  the  characteristics  of  bacteria  can  be 
radically  altered,  but  they  then  lose  those  special  traits  which  enable 
them  to  excite  disease  and  so  cease  to  exist  as  parasites  and  if  they 
have  lost  the  capacity  to  live  as  saprophytes  they  cease  to  exist  alto- 
gether. 

Attenuation. — As  just  stated  it  is  established  that  the  great  majority 
of  parasitic  bacteria  can  be  so  altered  by  being  grown  outside  the 
body,  and  especially  by  being  subjected  to  unfavorable  conditions, 
that  they,  while  morphologically  the  same,  lose  their  power  of  devel- 
oping in  the  body  and  of  producing  specific  poisons.  When  either  or 
both  of  these  properties  are  partially  destroyed  they  can  usually  be 
redeveloped ;  but  when  absolutely  lost  they  probably  cannot  be. 

The  recovery  of  toxin  production  is  brought  about  by  developing  the 
micro-organism  for  a  considerable  length  of  time  under  the  conditions 
best  suited  for  it.  The  recovery  of  the  ability  to  grow  in  the  body  of  any 
animal  species  is  brought  about  by  causing  the  germ  to  develop  in  a 
series  of  animals  of  ths  same  species  whose  resistance  has  been  over- 
come by  reducing  their  vitality  through  poisons,  heat,  cold,  etc.  Another 
method  is  to  accustom  the  micro-organism  to  the  animal's  body  by 
letting  it  remain  surrounded  by  the  animal  fluids  as  it  rests  in  a  per- 
vious capsule  in  the  peritoneal  cavity,  or  by  growing  it  in  unheated 
fresh  serum  or  blood  media. 

Chemical  Composition  of  Bacteria. — Qualitatively  considered,  the 
bodies  of  bacteria  consist  largely  of  water,  salts,  fats,  and  albuminous 
substances.  There  are  also  present,  in  smaller  quantities,  extractive 
substances  soluble  in  alcohol  and  in  ether.  Special  varieties  contain 
unusual  substances,  as  wax  and  cellules?  in  tubercle  bacilli.  Bacteria 
possess  the  capacity  in  a  high  degree  of  accommodating  their  chemical 
composition  to  the  variety  of  soil  in  which  they  are  growing.  The  same 
variety  of  bacteria  thus  varies  greatly  in  the  quantitative  estimation  of 
its  chemical  constituents.  Each  variety  yields  proteid  substances  pecu- 
liar to  itself,  as  shown  in  the  effects  produced  by  animal  inoculation. 
At  present  we  know  but  little  concerning  the  differentiation  of  these 
specific  substances.  This  subject  will  be  taken  up  in  detail  under 
bacterial  toxins,  etc.  According  to  Cramer  many  bacteria  contain 
amyloid  substances  which  give  a  blue  reaction  with  iodine.  True  cellu- 
lose has  been  found  in  some  bacteria,  and  also  large  quantities  of  a 
gelatinous  carbohydrate  similar  to  hemicellulose  have  been  obtained. 


40  PRINCIPLES  OF  BACTERIOLOGY 

Nuclein  is  found  frequently.  The  nuclein  bases — xanthin,  guanin,  and 
adenin — have  been  obtained  in  considerable  amounts.  There  is  a 
group  of  bacteria  which  contain  sulphur — viz.,  the  beggiatoa — and  an- 
other group,  the  cladothrix,  is  capable  of  separating  ferric  oxide  from 
water  containing  iron. 

Some  light  has  been  thrown  upon  the  chemical  composition  of  bacteria, 
quantitatively,  by  the  studies  of  Cramer,  though  so  far  only  a  few  species 
have  been  thoroughly  investigated.  The  percentage  of  water  contained 
in  bacteria  grown  on  solid  culture  media,  as  well  as  the  amount  of  ash, 
depends  largely  on  the  composition  of  the  media.  Thus  the  bacillus 
prodigiosus  when  grown  on  potato  contains  21.5  per  cent,  of  dry  residue 
and  2.7  per  cent,  of  ash;  when  cultivated  on  turnips  it  contains  12.6  per 
cent,  of  dry  residue  and  1.3  per  cent,  of  ash.  Besides  the  concentration 
of  the  culture,  its  temperature  and  age  also  influence  the  amount  of 
residue  and  ash  produced.  The  residue  varies,  moreover,  in  its  com- 
position in  the  same  species  under  the  influence  of  the  culture  media 
employed. 

It  appears  that  an  additional  quantity  of  peptone  in  the  culture  media 
tends  to  increase  the  percentage  of  nitrogenous  matter  in  the  bacillus, 
while  the  addition  of  glucose  decreases  it. 

Chemical  Substances  Necessary  for  the  Nutrition  of  Bacteria. — The 
majority  of  bacteria  are  easily  cultivated,  but  there  are  some  for 
which,  with  our  present  knowledge,  we  are  unable  to  produce  conditions 
suited  for  their  growth. 

All  bacterial  culture  media  must  contain  an  abundance  of  water; 
salts  are  also  indispensable,  and  there  must  be  organic  material  as  a 
source  of  carbon  and  nitrogen.  The  greater  number  of  important  bac- 
teria and  all  the  pathogenic  species  thrive  best  in  media  containing 
albuminoid  substances  and  of  a  slightly  alkaline  reaction  to  litmus. 
The  demands  of  bacteria  in  the  composition  of  the  culture  media  vary 
considerably.  There  are  some  species  of  water  bacteria,  for  instance, 
which  require  so  little  organic  material  that  they  will  grow  in  water  that 
has  been  twice  distilled.  A  certain  species  will  grow  abundantly  in 
water  containing  ammonium  carbonate  in  solution  and  no  other  source 
of  carbon  and  nitrogen.  This  shows  the  power  of  some  bacteria  of  pro- 
ducing cell  substance  from  the  simplest  materials — a  power  which 
belongs  to  the  higher  plants  which  obtain  their  nourishment  from  the 
air  through  their  chlorophyll  and  the  assistance  of  sunlight.  Few  bac- 
teria, however,  of  any  importance  in  medicine  are  so  easily  satisfied, 
though  there  are  many  species  which  are  able  to  develop  without  the 
presence  of  albumin  and  in  comparatively  simple  culture  media,  such 
as  the  culture  liquid  proposed  by  Uschinsky,  or  the  simpler  one  of  Voges 
and  Fraenkel,  which  consists  of  water,  1000;  sodium  chloride,  5; 
neutral  sodium  phosphate,  2;  ammonium  acetate,  6;  and  asparagin,  4. 
In  these  media  many  bacteria  grow  well. 

When  we  consider  in  detail  the  source  of  the  more  important  chemical 
ingredients  of  bacteria  we  find  that  their  nitrogen  is  most  readily  obtained 
from  diffusible  albuminoid  material  arid  less  easily  from  ammonium 


777 /•;  CLASSIFICATION  OF  BACTERIA  41 

compounds.  Their  carbon  they  derive  from  albumin,  peptone,  sugar, 
and  other  allied  carbohydrates;  glycerin,  fats,  and  other  organic  sub- 
stances. It  is  an  interesting  fact  that  even  compounds  which  in  con- 
siderable concentration  are  extremely  poisonous,  can,  when  in  sufficient 
dilution,  provide  the  necessary  carbon;  thus  some  derive  it  from  car- 
bolic acid  in  very  dilute  solutions. 

The  value  of  substances  as  a  source  of  nutrition  is  often  influenced 
by  the  presence  of  other  materials,  as,  for  instance,  the  value  of  asparagin 
is  increased  by  the  presence  of  sugars.  Further,  materials  from  which 
nitrogen  and  carbon  cannot  be  directly  obtained  still  become  assimilable 
after  being  subjected  to  the  influence  of  bacterial  ferments.  The  pro- 
found and  diverse  changes  produced  by  the  different  ferments  make  it 
almost  impossible  to  establish,  except  in  the  most  general  way,  the  nutri- 
tive value  of  any  mixture  for  a  large  number  of  bacteria  through  a  simple 
knowledge  of  its  chemical  composition.  The  special  culture  media, 
such  as  bouillon,  blood  serum,  etc.,  for  the  development  of  bacteria, 
will  be  dealt  with  in  a  later  chapter. 

Relation  of  Bacteria  to  Oxygen. — The  majority  absolutely  require 
oxygen  for  their  growth,  but  a  considerable  minority  fail  to  grow 
unless  it  is  excluded.  A  knowledge  of  this  latter  fact  we  owe  to  Pas- 
teur, who  divided  bacteria  into  aerobic  and  anaerobic.  Between  these 
two  groups  we  have  those  that  can  grow  either  with  or  without  the  access 
of  oxygen. 

Some  at  least  of  the  strict  anaerobic  bacteria  require  for  the  full  devel- 
opment of  their  life  functions  the  presence  of  fermentable  substances, 
such  as  sugars,  from  which  they  obtain  oxygen.  Among  bacteria  can 
be  found  all  gradations  between  those  bacteria  which  develop  only  in 
the  presence  of  oxygen  to  those  which  develop  only  in  its  absence.  In 
so  far  as  for  any  variety  the  amount  of  oxygen  present  is  unfavorable 
there  will  be  more  or  less  restriction  in  some  of  the  life  processes  of  these 
bacteria,  such  as  pigment  and  toxin  production,  spore  formation,  etc. 
It  has  also  been  found  that  some,  at  least,  of  the  aerobic  bacteria  can 
be  accustomed  to  grow  without  oxygen  and  that  some  of  the  anaerobics 
can  be  accustomed  to  grow  with  it  Free  oxygen  kills  the  vegetable  forms 
of  the  anaerobic  bacteria  in  a  few  hours,  but  hardly  injures  the  spores. 

Sulphur  and  phosphorus  are  two  important  foodstuffs  required  by 
bacteria.  Either  calcium  or  magnesium  and  sodium  or  potassium  are 
also  usually  required  for  bacterial  growth.  Iron  is  demanded  by  but 
few  varieties,  among  which  is  the  influenza  bacillus. 

When  we  consider  the  more  complex  culture  media,  either  those 
naturally  existing,  such  as  blood  serum,  or  those  created  by  us  for  the 
cultivation  of  bacteria,  we  find,  beyond  the  necessary  amount  of  soluble 
foodstuffs,  that  the  relative  proportion  of  each  form  and  the  total  con- 
centration are  of  great  importance.  It  is,  nevertheless,  true  that  very 
wide  differences  can  exist  with  but  slight  effect  upon  the  development 
of  many  bacteria,  the  development  of  the  bacteria  usually  ceasing 
through  the  accumulation  of  deleterious  substances  in  the  culture  media 
rather  than  through  food  exhaustion. 


42  PRINCIPLES  OF  BACTERIOLOGY 

Influence  of  Reaction  of  Media  upon  Growth. — The  reaction  of  the 
nutritive  media  is  of  very  great  importance.  Most  bacteria  grow  best  in 
those  that  are  slightly  alkaline  or  neutral  to  litmus.  Only  a  few  varieties 
require  an  acid  medium,  and  none  of  these  belong  to  the  parasitic  bac- 
teria. An  amount  of  acid  or  alkali  insufficient  to  prevent  the  develop- 
ment of  bacteria  may  still  suffice  to  rob  them  of  some  of  their  most 
important  functions,  such  as  the  production  of  poison.  The  different 
effect  upon  closely  allied  varieties  of  bacteria  of  a  slight  excess  of  acid 
or  alkali  is  sometimes  made  use  of  in  separating  those  which  may  be 
closely  allied  in  many  other  respects. 

Influence  of  One  Species  upon  the  Growth  of  Others. — The  influence  of 
one  species  upon  the  growth  of  another,  either  when  the  bacteria  grow 
together  or  follow  one  another,  is  very  marked.  The  development  of 
one  variety  of  bacteria  in  a  medium  causes  that  substance,  in  the  majority 
of  instances,  to  become  less  suitable  for  the  growth  of  other  bacteria. 
This  is  due  partly  to  the  impoverishment  of  the  foodstuff,  but  more  to 
the  production  of  chemical  substances  or  enzymes,  which  are  antagonistic 
not  only  to  the  growth  of  the  bacteria  producing  them,  but  to  many 
other  varieties  also ;  less  frequently  the  changes  produced  by  one  variety 
of  bacteria  in  the  foodstuff  are  favorable  for  some  other  form.  The 
pneumococcus  which  usually  develops  very  tiny  delicate  colonies  upon 
nutrient  agar,  grows  as  luxuriant  succulent  colonies  when  grown  with 
certain  bacilli. 

Temperature  Suitable  for  Growth. — For  the  growth  of  bacteria  a  suit- 
able temperature  is  absolutely  requisite.  For  different  varieties  the 
most  favorable  temperature  varies,  but  for  all  a  range  of  about  2.5°  C. 
above  or  below  this  most  favorable  point  covers  the  limits  for  their 
most  vigorous  growth.  Few  bacteria  grow  well  under  10°  C.  and  few 
over  40°  C.;  2°  C.  is  about  the  lowest  temperature  that  any  bacteria 
have  been  found  to  grow  and  70°  C.  the  highest. 

In  many  instances  the  temperature  of  the  soil  in  which  the  bacteria 
are  deposited  is  the  controlling  factor  in  deciding  whether  growth  will 
or  will  not  take  place.  Thus,  nearly  all  parasitic  bacteria  require  a  tem- 
perature near  that  of  the  body  for  their  development,  while  many  sapro- 
phytic  bacteria  can  grow  only  at  much  lower  temperatures.  Bacteria 
when  exposed  to  lower  temperature  than  suffices  for  their  growth,  while 
having  their  activities  decreased,  are  not  otherwise  injured  unless  actually 
frozen;  while  exposure  to  higher  temperatures  than  allows  of  growth 
destroys  the  life  of  the  bacteria.  The  relations  of  the  temperature 
to  bacterial  life  and  death  will  be  dealt  with  more  fully  in  the  next 
chapter. 


CHAPTER    IV. 

EFFECT  OF  TEMPERATURE   UPON  THE  GROWTH  OF  BACTERIA. 

Ix  judging  the  effect  on  bacteria  of  heat  as  well  as  other  agents  we 
have  to  note  the  important  fact  that  different  species  are  differently 
influenced  by  the  same  substance.  Some  bacteria  live  under  conditions 
which  would  destroy  others,  and  they  vary  among  themselves  in  their 
powers  of  resistance  to  influences  which  are  deleterious  to  all. 

Further,  any  species  of  bacteria  will  resist  better  when  under  favor- 
able conditions  than  under  unfavorable  ones.  Bacteria  also  in  recent 
cultures  withstand  injury  better  than  those  in  old  cultures,  so  long  as 
they  have  not  entered  into  the  spore  form.  According  to  the  amount  of 
injury  they  have  suffered,  bacteria  may  be  inhibited  in  some  of  their 
functions  or  they  may  be  totally  destroyed. 

Bacteria  Divided  According  to  the  Temperatures  at  which  they  Grow 
Best. — Some  form  of  bacterial  life  is  possible  within  the  limits  of  0°  and 
70°  C.  There  are  some  species,  however,  which  grow  at  the  lower  and 
others  at  the  upper  limit  of  these  temperatures.  The  maximum  and 
minimum  temperature  for  each  individual  species  lies  from  10°  to  30°  C. 
apart.  Bacteria  have  been  classified  according  to  the  temperatures  at 
which  they  develop,  as  follows : 

PSYCHKOPHILIC  BACTERIA. — Minimum  at  0°  C.,  optimum  at  15°  to 
20°  C.,  maximum  at  about  30°  C.  To  this  class  belong  many  of  the 
water  bacteria,  such  as  the  phosphorescent  bacteria  in  sea-water. 

MESOPHILIC  BACTERIA. — Minimum  at  5°  to  25°  C.,  optimum  at 
37°  C.,  maximum  at  about  43°  C.  To  this  class  belong  all  patho- 
genic bacteria. 

THERMOPHILIC  BACTERIA. — Minimum  at  25°  to  45°  C.,  optimum  at 
50°  to  55°  C.,  maximum  at  60°  to  70°  C.  This  class  includes  a  number 
of  soil  bacteria  which  are  almost  exclusively  spore-bearing  bacilli.  They 
are  also  found  widely  distributed  in  feces. 

By  carefully  elevating  or  reducing  the  temperature  the  limits  within 
which  a  variety  of  bacteria  will  grow  can  be  altered.  Thus,  the  anthrax 
bacillus  was  gradually  made  to  accommodate  itself 'to  a  temperature  of 
42°  C.,  and  pigeons,  which  are  comparatively  immune  to  anthrax,  partly 
on  account  of  their  high  body  temperature  (42°  C.),  when  inoculated  with 
this  anthrax  succumbed  to  the  infection.  Another  culture  accustomed 
to  a  temperature  of  12°  C.  killed  frogs  kept  at  12°  C.  We  have  culti- 
vated a  very  virulent  diphtheria  bacillus  so  that  it  will  grow  at  43°  C. 
and  produce  strong  toxin. 

Effect  of  Low  Temperature. — Bacterial  growth  is  retarded  by  tempera- 
tures lower  than  their  optimum,  although  the  bacteria  are  not  otherwise 


44  PRINCIPLES  OF  BACTERIOLOGY 

injured.  Indeed,  it  is  the  usual  custom  in  laboratories  to  preserve 
bacteria  which  die  readily  (such  as  streptococci)  by  keeping  them  in  the 
refrigerator  at  about  4°  to  6°  C.,  after  cultivation  for  two  days  at  30°  C., 
as  a  means  for  retaining  their  vitality  without  repeated  transplantation. 
Temperatures  even  far  under  0°  C.  are  only  slowly  injurious  to  bacteria, 
different  species  being  affected  with  varying  rapidity.  This  has  been 
demonstrated  by  numerous  experiments  in  which  they  have  been  exposed 
for  hours  in  a  refrigerating  mixture  at  — 18°  C.  They  have  even  been 
subjected  to  a  temperature  of  — 175°  C.  by  immersing  them  in  liquid 
air  kept  in  an  open  tube  for  two  hours,  arid  15  to  80  per  cent,  were  found 
to  still  grow  when  placed  in  favorable  conditions.  We  found  about 
10  per  cent,  of  typhoid  bacilli  alive  after  thirty  minutes'  exposure  to 
this  low  temperature.  Staphylococci  were  more  resistant.  Spores  were 
scarcely  killed  at  all. 

Effect  of  High  Temperatures. — Temperatures  from  5°  to  10°  C.  over 
the  optimum  affect  bacteria  injuriously  in  several  respects.  Varieties 
are  produced  of  diminished  activity  of  growth,  the  virulence  and  the 
property  of  causing  fermentation  are  decreased,  and  the  power  of  spore 
formation  is  gradually  lost.  These  effects  may  predominate  either  in 
one  or  the  other  direction. 

If  the  maximum  temperature  is  exceeded  the  organism  dies;  the 
thermal  death  point  for  the  psychrophilic  species  being  about  37°  C., 
for  the  mesophilic  species  about  45°  to  55°  C.,  and  for  the  thermophilic 
species  about  75°  C.  There  are  no  non-spore  bearing  bacteria  which 
when  moist  are  able  to  withstand  a  temperature  of  100°  C.  even  for  a 
few  minutes.  A  long  exposure  to  temperatures  between  60°  and  80°  C. 
has  the  same  result  as  a  shorter  one  at  the  higher  temperatures.  Ten 
minutes'  exposure  to  moist  heat  will  at  60°  C.  kill  the  cholera  spirillum, 
the  streptococcus,  the  typhoid  bacillus,  and  the  gonococcus,  and  at 
70°  C.  the  staphylococcus,  the  latter  being  among  the  most  resistant 
of  the  pathogenic  organisms  which  have  no  spores. 

Effect  of  Dry  Heat. — When  micro-organisms  in  a  desiccated  condition 
are  exposed  to  the  action  of  heated  dry  air  the  temperature  required  for 
their  destruction  is  much  above  that  required  when  they  are  in  a  moist 
condition  or  when  they  are  exposed  to  the  action  of  hot  water  or  steam. 
A  large  number  of  pathogenic  and  non-pathogenic  species  are  able  to 
occasionally  resist  a  temperature  of  over  100°  C.  dry  heat  for  an  hour.  In 
any  large  number  of  bacteria  a  few  are  always  more  resistant  than  the 
majority.  A  temperature  of  120°  to  130°  C.  dry  heat  maintained  for  one 
and  a  half  hours  will  destroy  all  bacteria,  in  the  absence  of  spores. 

Resistance  of  Spores  to  Heat. — Spores  are  far  more  resistant  to  all 
injurious  influences  than  vegetative  forms.  They  retain  their  power 
of  germination  for  years  without  either  nourishment  or  water,  and  are 
much  more  indifferent  to  the  action  of  gases  than  bacilli,  the  spores  of 
the  anaerobic  species  being  especially  resistant  to  the  action  of  oxygen. 
Spores  possess  a  great  power  of  resistance  to  both  moist  and  dry  heat. 
Dry  heat  is  comparatively  well  borne,  many  spores  resisting  a  tempera- 
ture of  over  130°  C.  for  as  long  as  three  hours.  Exposed  to  150°  C.  for 


EFFECT  OF  TEMPERATURE  UPOX  THE  GROWTH  OF  BACTERIA  45 

one  hour,  practically  all  spores  are  killed.  Moist  heat  at  a  temperature 
of  100°  C.,  either  boiling  water  or  free-flowing  steam,  destroys  the 
spores  of  known  pathogenic  bacteria  within  fifteen  minutes;  certain 
non-pathogenic  species,  however,  resist  this  temperature  for  hours.  The 
spores  of  a  bacillus  from  the  soil  required  five  and  a  half  to  six  hours' 
exposure  to  streaming  steam  for  their  destruction.  They  were  destroyed, 
however,  by  exposure  for  twenty-five  minutes  in  steam  at  113°  to  116°  C. 
and  in  two  minutes  at  127°  C. 

The  resistance  of  spores  to  moist  heat  is  tested  by  suspending  threads, 
upon  which  the  spores  have  been  dried,  in  boiling  water  or  steam.  The 
threads  are  removed  from  minute  to  minute  and  laid  upon  agar  or  in 
broth,  which  is  then  placed  at  a  suitable  temperature. 

Practical  Use  of  Heat  Disinfection. — In  the  practical  application  of 
steam  for  disinfecting  purposes  it  must  be  remembered  that  while  moist 
steam  under  pressure  is  more  effective  than  streaming  steam  it  is  scarcely 
necessary  to  give  it  the  preference,  in  view  of  the  fact  that  most  known 
pathogenic  bacteria  produce  no  spores  and  the  spores  of  the  few  that  do 
develop  them  are  quickly  destroyed  by  the  temperature  of  boiling  water, 
and  also  that  "superheated"  steam  is  less  effective  than  moist  steam. 
When  confined  steam  in  pipes  is  "superheated"  after  its  generation  it  has 
about  the  same  germicidal  power  as  hot,  dry  air  at  the  same  temperature. 
Esmarch  found  that  anthrax  spores  were  killed  in  streaming  steam  in 
four  minutes,  but  were  not  killed  in  the  same  time  by  superheated  steam 
at  a  temperature  of  114°  C.  It  should  also  be  remembered  that  dry  heat 
has  but  little  penetrating  powder,  and  that  even  steam  requires  time  to 
pass  through  heavy  goods.  Koch  and  Wolff hugel  found  that  registering 
thermometers  placed  in  the  interior  of  folded  blankets  and  packages 
of  various  kinds  did  not  show  a  temperature  capable  of  killing  bacteria 
after  three  hours'  exposure  in  a  dry  hot-air  oven  at  133°  C.  and  over. 
We  have  often  put  a  piece  of  ice  in  the  middle  of  several  mattresses  and 
recovered  it  after  exposing  the  goods  to  an  atmosphere  of  live  steam  for 
ten  minutes. 

FRACTIONAL  STERILIZATION. — Certain  nutrient  media,  such  as  blood- 
serum  and  the  transudates  of  the  body  cavities,  as  well  as  certain  fluid 
foodstuffs,  need  at  times  to  be  sterilized,  and  yet  cannot  be  subjected 
to  temperatures  high  enough  to  kill  spores  without  suffering  injury. 
The  property  of  spores,  when  placed  under  suitable  conditions,  to  germi- 
nate into  the  non-spore  bearing  form,  is  here  taken  advantage  of  by 
heating  the  fluids  up  to  55°  to  70°  C.  for  one  hour  on  each  of  six  con- 
secutive days,  and  keeping  the  fluid  at  about  20°  C.  during  the  intervals. 
By  this  means  we  kill,  upon  each  exposure,  all  bacteria  in  the  vegetative 
form,  and  allow,  during  the  intervals,  for  the  development  of  any  still 
remaining  in  the  spore  stage,  or  which  have  reproduced  spores,  to 
change  again  into  the  vegetative  form.  Experience  has  shown  that, 
with  but  few  exceptions,  an  exposure  for  six  consecutive  days  will  com- 
pletely sterilize  the  fluids  so  exposed. 

With  the  usual  culture  media  a  temperature  of  100°  C.  for  twenty 
minutes  does  little  or  no  harm  while  one  of  115°  C.  is  deleterious.  With 


46  PRINCIPLES  OF  BACTERIOLOGY 

heating   to   100°   C.   an   exposure   on  three    consecutive  days,  and  to 
115°  C.  one  or  two  suffices. 

PASTEURIZATION. — It  is  sometimes  undesirable  to  expose  food,  such 
as  milk,  to  such  a  temperature  as  will  destroy  spores,  because  of  the 
deleterious  effects  upon  food  values  of  such  high  temperatures,  and  yet 
where  a  partial  destruction  is  necessary.  In  these  cases  we  heat 
the  foodstuffs  for  from  five  to  thirty  minutes  to  such  a  temperature 
(70°  C.)  as  will  kill  most  of  the  bacteria  in  the  vegetative  form,  but 
allow  the  spores  to  remain  alive.  Even  this  amount  of  sterilization  kills 
about  90  to  98  per  cent,  of  the  bacteria.  The  exposure  to  this  degree 
of  heat  alters  the  chemical  composition  of  the  milk  but  little. 


CHAPTER  V. 

THE  MATERIALS  AND  METHODS  USED  IN  THE  CULTIVATION 

OF  BACTERIA. 

THE  methods  for  the  artificial  cultivation  of  bacteria  are  of  funda- 
mental importance  in  bacteriology.  The  study  of  the  characteristics 
of  any  bacterium  requires  that  it  be  examined  growing  apart  from  all 
others.  In  order  to  separate  one  species  from  others  and  to  study  its 
morphological,  biochemical,  and  cultural  characteristics  we  have  to 
prepare  a  number  of  sterile,  solid,  and  liquid  media  and  employ  them 
in  various  technical  ways.  Before  we  can  get  a  suitable  growth  of  any 
special  variety  of  bacteria,  we  must  have  the  substances  necessary  for 
their  growth  present  in  the  proper  proportion  and  concentration.  Dif- 
ferent species  of  bacteria  require  very  different  foodstuffs,  so  that  for 
each  kind  the  proper  food  must  be  found  through  experimentation. 

Preparation  of  Culture  Media. — The  most  commonly  used  media  have 
as  their  basis  the  watery  extract  of  meat  and  peptone.  The  addi- 
tion to  this  by  Koch  of  gelatin  gave  us  a  transparent  solid  medium 
which  had,  however,  the  objection  of  melting  below  the  temperature 
required  for  the  growth  of  many  pathogenic  bacteria.  Another  sub- 
stance of  vegetable  origin  (agar)  was  found,  which  melted  just  below  the 
boiling  point  of  water.  This  has  been  substituted  for  gelatin  whenever 
we  desire  to  grow  bacteria  at  temperatures  above  20°  C.  or  desire  other 
characteristics  of  the  agar  media. 

PREPARATION  OF  MEAT  IXFUSIOX. — One  pound  (500  grams)  of  finely 
chopped,  fresh,  lean  meat  is  macerated  in  1000  c.c.  of  water  and  put  in 
an  ice-chest  for  from  eighteen  to  twenty-four  hours;  or  it  may  be  warmed 
at  a  temperature  not  exceeding  60°  C.  for  one  hour.  Any  fat  present  is 
skimmed  off.  The  last  traces  can  be  removed  by  stroking  the  surface 
with  filter-paper.  The  infusion  is  now  strained  through  a  fine  cheese- 
cloth into  a  flask,  and  the  remaining  meat  placed  in  a  cloth  and  squeezed 
by  hand  or  in  a  press.  The  resulting  fluid  contains  the  soluble  albumin, 
the  soluble  salts,  extractives,  and  coloring  matter  of  the  meat.  This 
meat  extract  is  then  exposed  to  live  steam,  either  without  pressure  in  the 
Arnold  steam  sterilizer  (Fig.  21)  for  thirty  minutes,  or,  if  the  changes 
produced  by  a  temperature  of  110°  to  115°  C.  are  not  objectionable,  in 
the  autoclave  at  one  atmosphere  of  pressure  for  fifteen  minutes,  or 
boiled  over  a  free  flame  for  ten  minutes.  During  this  process  all  albumins 
are  coagulated.  While  still  hot  the  fluid  is  filtered  through  filter-paper 
or  through  absorbent  cotton,  and  the  reaction  is  tested  and  sufficient 
normal  hydrochloric  acid  solution  or  sodium  hydroxide  added  to  give 
it  the  desired  reaction,  which  is  for  most  bacteria  slightly  alkaline  to 
litmus.  If  in  the  boiling  there  has  been  any  evaporation,  sufficient 


48 


PRINCIPLES  OF  BACTERIOLOGY 


FIG.  21 


water  is  added  to  bring  the  fluid  up  to  its  original  bulk.  If  the  fluid  is 
clear  it  is  put  into  flasks  and  tubes  and  sterilized;  if  not  clear,  the  white 
of  one  or  two  eggs  is  added  to  the  fluid  after  cooling  it  down  to  about 
55°  C.  After  thoroughly  mixing  the  eggs,  the  bouillon  is  boiled  briskly 
for  a  few  minutes,  its  reaction  adjusted,  and  then  again  filtered  and  dis- 
tributed in  flasks  and  put  in  the  Arnold 
sterilizer  for  one  hour  on  each  of  three 
consecutive  days,  or  in  the  autoclave 
for  twenty  minutes  for  sterilization. 
Instead  of  meat  2  to  4  grams  of  Lie- 
big's  or  some  other  meat  extract  are 
added  to  each  litre  of  water.  For  some 
purposes  the  extract  is  as  good  as  the 
fresh  meat,  but  for  others  it  is  inferior. 
This  infusion  contains  very  little  albu- 
minous matter,  and  consists  chiefly  of 
the  soluble  salts  of  the  muscle,  certain 
extractives,  and  any  slight  traces  of 
soluble  proteid  not  coagulated  by  heat. 
It  is  not,  therefore,  a  suitable  medium 
for  most  bacteria. 

We  use  this  as  a  basis  for  the  fol- 
lowing more  useful  media : 

BOUILLON  MEDIA. — These  consist 
of  meat  infusion  plus  certain  sub- 
stances. 

(a)  Peptone  or  Nutrient  Bouillon.- — 
This  has  the  composition:  meat  infu- 
sion, 1000  c.c.;  sodium  chloride,  5  grams;  peptone  (Witte),  10  grams. 
Warm  moderately  and  stir  until  the  ingredients  are  dissolved,  then 
boil  for  thirty  minutes  in  the  Arnold  sterilizer  or  the  autoclave  and 
treat  as  in  making  meat  infusion.  For  the  careful  study  of  bacteria 
the  exact  reaction  of  the  media  should  be  carefully  determined.  For 
this  purpose  standard  solutions  are  used  with  phenolphthalein  or 
litmus  as  an  indicator.  This  subject  will  be  taken  up  in  detail  at  the 
end  of  the  chapter.  For  water  bacteria  sodium  chloride  is  omitted  and 
the  reaction  is  made  + 1  per  cent. 

(b)  Sugar  Bouillon. — To  the  peptone  broth  after  its  completion  1  to  2 
per  cent,  of  glucose,  lactose,  saccharose,  or  other  sugars  is  added.    No 
more  boiling  than  necessary  to  sterilize  should  be  used  after  the  addition 
of  the  sugars,  since  they  become  altered  by  heat.     Temperatures  higher 
than  100°  C.  should  never  be  employed.      These  media  are  used  to 
determine  the  effect  of  bacteria  upon  the  different  sugars. 

(c)  Glycerin-peptone  Bouillon. — After   filtration,  3  to  5  per  cent,  of 
glycerin  is  added  to  the  peptone  bouillon  and  the  whole  again  sterilized. 
This  medium  is  used  especially  for  the  growth  of  the  tubercle  bacilli. 

(d)  Mannite-peptone  Bouillon. — This  is  prepared  by  adding  1  per  cent, 
mannite  to  the  peptone  bouillon.    It  is  used  especially  in  differentiating 


Arnold  steam  sterilizer. 


CULTIVATION  OF  BACTERIA  49 

the  varieties  of  dysentery  bacilli,  some  fermenting  mannite  and  others 
not.     In  careful  work  the  bouillon  must  be  rendered  sugar  free. 

PEPTONE  SOLUTION  (Dunham's). — This  is  a  simple  1  to  2  per  cent, 
solution  of  peptone  in  tap  or  distilled  water  to  which  5  per  cent,  of 
sodium  chloride  is  added.  The  peptone  and  sodium  chloride  are  dis- 
solved by  heating.  The  fluid  is  filtered,  placed  in  tubes,  and  sterilized. 
The  reaction  is  slightly  alkaline  to  litmus  and  suitable  for  most  pur- 
poses. It  can  be  altered  or  standardized  if  desired. 

(a)  Sugar-peptone  Solution,  etc. — The  various  sugars,  mannite,  inulin, 
and   glycerin  are  added  to    the    peptone  solution   just  as   previously 
described  for  bouillon. 

(b)  Sugar-free  Bouillon. — A  quantity  of  a  culture  of  bacillus  coli  or 
of  bacillus  lactis  aerogenes  is  added  to  the  meat  extract  and  incubated 
at  37°  for  twenty-four  hours.     The  acidity  is  neutralized,  peptone  and 
salt  added,  and  treated  as  described  under  bouillon. 

GELATIN  MEDIA. — These  are  simply  the  foregoing  bouillon  and 
peptone  media  to  which  gelatin  is  added  as  follows:  To  the  bouillon 
already  prepared  as  described  add  10  per  cent,  of  sheet  gelatin  and 
neutralize.  Add  the  whites  of  two  eggs  for  each  litre,  and  boil  for  a  few 
minutes.  Filter,  place  in  tubes  or  flasks,  and  sterilize.  Instead  of  add- 
ing gelatin  to  bouillon  already  prepared,  it  may  be  added  to  the  meat 
infusion  at  the  same  time  the  peptone  and  salt  were  added  in  preparing 
nutrient  bouillon  as  just  described.  Different  preparations  of  gelatin 
differ  greatly  as  to  their  melting  point.  Boiling  lowers  the  melting 
point,  so  that  heat  should  not  be  applied  any  longer  than  necessary. 
The  melting  point  of  different  samples  of  nutrient  gelatin  varies  between 
20°  to  27°  C.  The  "gold-label"  gelatin  is  employed. 

AGAR  MEDIA. — These  are  the  various  bouillon  and  peptone  media 
to  which  1  to  2  per  cent,  of  agar  are  added.  In  order  to  lessen  the  effect 
of  heat  on  sugars  simple  nutrient  agar  is  first  prepared  and  then  the 
sugar  added.  They  are  prepared  by  adding  to  stock  bouillon  1  or  2 
per  cent.,  as  desired,  of  thread  agar,  melting  it  by  placing  over  a  free 
flame  or  in  the  autoclave  or  steam  sterilizer.  When  the  agar  is  brought 
into  solution  over  a  free  flame  there  may  be  considerable  loss  of  fluid  by 
evaporation.  This  should  be  compensated  for  by  adding  additional 
water  before  boiling.  Agar  may  be  added  directly  to  the  meat  infusion 
along  with  the  peptone  and  salt.  Indeed,  this  is  an  advantage,  as  agar- 
agar  is  very  difficult  to  bring  into  solution,  and  is  not  injured  in  the  least 
by  prolonged  boiling.  The  agar  may  be  added  to  water  alone  in  double 
the  amount  finally  desired.  To  this  is  added  an  equal  quantity  of  nu- 
trient broth,  which  is  also  double  its  usual  strength.  Glycerin  agar  is 
simply  nutrient  agar  plus  3  to  6  per  cent,  of  glycerin.  It  is  added  to  the 
hot  nutrient  agar  just  previous  to  putting  it  in  the  flasks.  Nutrient  agar 
begins  to  thicken  at  a  fairly  high  temperature,  and  should  be  filtered  as 
hot  as  possible.  When  small  amounts  are  made  it  is  well  to  place  the 
filter  and  receiving  flask  in  the  sterilizer  while  filtering. 

MILK. — This  fluid  is  a  good  culture  medium  for  most  pathogenic 
bacteria.  It  should  be  obtained  as  fresh  as  possible,  so  that  but  little 


50  PRINCIPLES  OF  BACTERIOLOGY 

bacterial  change  has  occurred.  It  is  first  put  in  the  ice-chest  for 
twelve  hours  to  allow  the  cream  to  rise.  The  milk  is  then  siphoned  off 
from  below  the  cream  into  a  flask  and  its  reaction  tested.  After  correc- 
tion it  is  put  in  tubes  or  flasks  and  sterilized.  If  acid  to  phenolphthalein, 
normal  sodium  hydrate  should  be  added  to  make  it  — 1  per  cent. 

LITMUS  MEDIA. — When  it  is  desirable  to  determine  whether  bacteria 
produce  in  their  growth  acid  or  alkali  from  one  or  more  of  the  constit- 
uents of  the  media  litmus  is  frequently  added.  To  prepare  the  litmus 
solution  take  the  lump  litmus,  powder  finely,  and  boil  with  distilled 
water  so  that  a  saturated  solution  is  obtained.  Filter  and  then  boil  for 
thirty  minutes  on  two  successive  days.  The  litmus  solution  is  added 
to  the  neutral  media  in  sufficient  quantity  to  give  the  desired  depth  of 
color.  The  less  heating  that  is  done  after  mixing  the  better  the  results. 
Merck's  purified  litmus  in  1  per  cent,  aqueous  solution  should  be  used 
in  careful  work. 

PETRUSKY'S  LITMUS  WHEY  (as  modified  by  Durham). — Fresh  milk 
is  slightly  warmed  and  clotted  by  means  of  essence  of  rennet.  The 
whey  is  strained  off  and  the  clot  is  hung  up  to  drain  in  a  piece  of  muslin. 
The  whey,  which  is  somewhat  turbid,  is  then  cautiously  neutralized 
with  4  per  cent,  citric  acid  solution,  neutral  litmus  being  used  as  an 
indicator.  When  it  gives  a  good  neutral  violet  color  with  the  litmus  it 
is  heated  at  100°  C.  for  one  hour;  thereby  nearly  the  whole  proteid  is 
coagulated.  It  is  thus  filtered  clear,  and  neutral  litmus  is  added  to  a 
convenient  color  for  observation. 

NEUTRAL  RED. — This  dye  is  added  to  the  peptone  and  bouillon- 
sugar  media  to  the  amount  of  1  to  5  per  cent,  of  a  concentrated  solution. 
Its  reduction  by  the  growth  of  bacteria  is  a  valuable  point  in  differentia- 
tion. 

NITRATE  BOUILLON.  —  Dissolve  10  grams  of  peptone  in  1  litre  of 
spring  or  tap  water  and  add  0.02  grams  of  potassium  nitrate  (which 
is  free  of  nitrites).  This  is  placed  in  test-tubes  and  sterilized. 

POTATOES. — Potatoes  are  used  for  some  special  purposes.  The 
potatoes  may,  after  thorough  scrubbing  and  removal  of  "eyes/7  be 
soaked  in  bichloride  of  mercury  (1 :1000)  for  twenty  minutes,  and  then 
sterilized  on  three  consecutive  days  for  one-half  hour  in  the  steam  ster- 
ilizer. To  use  they  are  cut  in  thick  slices  and  put  in  deep  Petri  dishes. 
For  more  careful  work  the  potatoes  are  first  cut  into  proper  sizes  for 
tubes,  and  then  soaked  for  twelve  hours  in  running  water  to  remove  the 
acidity. 

BLOOD  AND  BLOOD-SERUM  MEDIA,  (a)  Fresh  Blood  Media. — These 
are  made  by  streaking  sterile  defibrinated  or  fresh  human,  rabbit,  or  other 
blood  over  nutrient  agar  contained  in  tubes  or  dishes.  Sometimes  fresh 
blood  is  added  to  fluid  nutrient  agar  at  40°  C.  or  to  bouillon  and  a 
mixture  thus  obtained.  Media  made  with  fresh  blood  contains  not  only 
the  haemoglobin,  but  also  intact  red  blood  cells.  Blood  media  are  used 
for  the  growth  of  the  influenza  bacillus,  for  pneumococci  and  other 
bacteria,  and  for  the  observation  of  the  reduction  of  the  haemoglobin 
by  the  growth  of  certain  bacteria. 


CULTIVATION  OF  BACTERIA 


51 


(b)  Heated  Blood  Media. — The  clot  containing  the  red  cells,  after  the 
separation  of  the  serum,  is  broken  up  and  added  to  bouillon  and  heated 
to  80°  to  90°  C.  This  makes  a  muddy  fluid  which  is  fitted  only  for 
the  development  of  bacteria  where  no  observation  of  their  growth  is 
required. 

BLOOD-SERUM  MEDIA.  ASCITIC  OR  PLEURITIC  FLUID. — Blood  serum 
may  be  sterilized  by  fractional  sterilization  and  remain  fluid,  or  it  may 
be  rendered  solid  by  the  degree  of  heat  used  in  sterilizing.  The  blood 
is  obtained  from  an  ox,  horse,  sheep,  dog,  or  rabbit  and  collected  into 
jars,  flasks,  or  tubes.  When  it  is  to  be  used  in  a  fluid  state  it  should  be 
drawn  in  an  aseptic  manner  into  a  flask  from  a  vein  by  means  of  a 
sterile  cannula  and  rubber  tube.  When  it  is  to  be  solidified,  less  care  is 
necessary.  It  is  here  sufficient  to  catch  the  blood  from  the  cut  artery 
or  vein  into  sterile  jars  or  tubes.  To  facilitate  clotting  it  is  well  to  have 
in  the  jar  or  tube  something  for  the  clot  to  contract  around. 

Loeffler's  Blood  Serum. — Three  parts  of  a  calf's  or  sheep's  blood  is 
mixed  with  one  part  of  neutral  peptone  bouillon  containing  1  per  cent, 
of  glucose.  The  serum  mixture  is  run  into  tubes,  which  are  plugged  and 
then  placed  in  a  slanting  position  in  the  serum  inspissator. 

Serum  may  be  solidified  and  still  remain  translucent  at  a  temperature 
of  76°  C.,  but  when  heated  to  a  higher  degree  a  more  definite  coagula- 
tion takes  place,  and  the  medium  becomes  opaque.  Care  must  be 
taken  in  coagulating  blood  serum  at  the  higher  temperature  to  run  the 
temperature  up  slowly,  and  not  to  heat  above  95°  C.  until  the  serum 
has  firmly  coagulated;  for,  unless  these  precautions  are  taken,  ebullition 
is  likely  to  occur,  which  will  lead  to  the  formation  of  bubbles  and  an 
unevenness  of  the  surface  upon  which  growth  is  to  be  obtained  and 
studied.  Serum  may  be  solidified 
at  the  temperature  mentioned  in  an 
incubator,  water-oven,  or  even  in 
an  Arnold  sterilizer  with  the  top 
covered  by  a  cloth  instead  of  the 
usual  lid,  and  when  coagulated 
firmly  (90°  C.)  the  tubes  and  their 
contents  may,  on  the  following  day, 
be  sterilized  in  streaming  steam 
at  100°  C.  without  danger  of  the 
subsequent  formation  of  bubbles. 
Koch's  serum  coagulator  (Fig.  22) 
is,  however,  the  most  convenient 

apparatus.  Some  bacteriologists  prepare  the  tubes  of  solidified  serum 
in  the  autoclave,  gradually  increasing  the  temperature  to  110°  C.  This 
is  a  very  rapid  and  convenient  method.  It  has  seemed  to  us,  however, 
that  the  high  temperature  injured  the  medium  somewhat. 

Alkaline  Blood  Serum. — To  each  100  c.c.  of  blood  serum  add  1  to  1.5 
c.c.  of  a  10  per  cent,  solution  of  sodium  hydrate.  Treat  as  Loeffler's 
serum.  This  will  give  a  solid,  clear  medium  consisting  chiefly  of  alkali 
albumin. 


52  PRINCIPLES  OF  BACTERIOLOGY 

Serum-bouillon  Media  (M  armorek's  Media) : 

1.  Human  serum,  2  parts;  nutrient  bouillon,  1  part. 

2.  Ascitic  or  pleuritic  fluid,  1  part;  nutrient  bouillon,  2  parts. 

3.  Horse  serum,  1  to  2  parts;  nutrient  bouillon,  1  to  2  parts. 
These  media  were  first  used  extensively  by  Marmorek  in  cultivating 

streptococci.  The  ascitic  fluid  bouillon  has  been  found  by  Williams 
to  be  of  great  use  in  enriching  cultures  of  diphtheria  bacilli.  It  is  also 
the  best  medium  for  the  growth  of  pneumococci,  streptococci,  and  many 
other  pathogenic  bacteria. 

Serum-water  Media  (Hiss'  Serum  Media). — When  diluted  with  2  to 
10  parts  of  water,  many  sera  can  be  steamed  without  coagulating. 

1.  Ox  serum,  1  part;  distilled  water,  2  parts;  normal  sodium  hydrate, 
0.1  per  cent. 

2.  The  same,  with  inulin  1  per  cent,  substituted  for  the  sodium  hydrate. 
For  the  sterilization  of   undiluted  fluid  serum  and  of  ascitic   and 

pleuritic  fluids,  it  is  requisite  that  it  be  exposed  to  a  temperature  of 
from  62°  to  66°  C.  for  one  hour  on  each  of  six  consecutive  days.  The 
best  apparatus  for  obtaining  and  maintaining  this  temperature  (about 
65°  C.)  is  a  small  and  well-regulated  incubator  or  chamber  surrounded 
by  a  water  space,  into  which  the  tubes  and  flasks  containing  serum  are 
to  be  put  each  day,  and  in  which  they  are  to  be  left  for  the  prescribed 
time  after  having  been  warmed  to  the  desired  temperature. 

Serum  may  be  preserved  by  placing  it  in  flasks  which,  after  the  addi- 
tion of  5  per  cent,  of  chloroform,  are  sealed.  When  it  is  to  be  used  it  is 
filled  into  sterilized  culture  (test)  tubes  and  sterilized  by  exactly  the 
same  methods  as  are  employed  in  sterilizing  fresh  serum.  The  chloro- 
form, being  volatile,  tends  to  disappear  at  ordinary  temperatures,  but 
is  quickly  and  surely  driven  off  at  the  temperatures  used  in  sterilizing. 

Serum  may  be  efficiently  sterilized,  when  great  care  is  used,  by  pass- 
ing it  through  a  well-tested  Pasteur  filter,  under  pressure.  When  so 
treated  the  fluid  is  very  clear  and  light-colored. 

Important  media  used  for  special  varieties  of  bacteria  will  be  noted 
in  the  chapters  devoted  to  them. 

Reaction  of  Culture  Media. — The  reaction  of  media  is  a  matter  of 
the  greatest  importance,  since  slight  variations  will  often  aid  or  inhibit 
the  growth  of  bacteria  and  also  produce  marked  differences  in  the 
microscopic  and  macroscopic  characters  of  a  growth.  Hence  it  is  impor- 
tant to  work  with  media  whose  reaction  is  accurately  known,  so  that 
necessary  variations  or  uniformity  can  afterward  be  attained 

Formerly  it  was  customary  to  use  litmus  as  the  indicator  in  neutral- 
izing media,  adding  normal  soda  or  hydrochloric  acid  solution  until  the 
red  litmus  turned  slightly  blue,  or  the  blue  litmus  just  a  tinge  less  blue. 
This  was  considered  the  neutral  point.  This  is  still  a  satisfactory  method 
for  those  who  are  only  going  to  cultivate  the  common  pathogenic  bacteria 
for  diagnostic  purposes  or  for  the  development  of  toxin.  Most  parasitic 
bacteria  which  grow  at  all  on  artificial  culture  media  develop  best  in 
them  when  they  have  a  neutral  or  slightly  alkaline  reaction  to  litmus. 
If  a  certain  alkalinity  is  desired  a  definite  number  of  cubic  centimetres 


CULTIVATION  OF  BACTERIA  53 

of  normal  soda  solution  can  be  added  for  each  litre  of  neutral  media; 
if  an  acidity  is  desired,  normal  hydrochloric  acid  solution  is  added. 

Many  bacteriologists  consider  that  litmus  is  not  delicate  enough  to 
be  entirely  satisfactory,  especially  when  experiments  are  to  be  reported 
or  exactly  repeated.  This  objection  is  made  chiefly  by  those  investi- 
gating water  bacteria  who  are  watching  cultural  and  biochemical  charac- 
teristics in  simple  peptone-beef  media.  For  these  purposes  phenol- 
phthalein  has  been  generally  selected.  It  is  of  great  importance  to  re- 
member that  different  indicators  not  only  differ  in  delicacy,  but  that  they 
react  differently  to  different  substances.  A  medium  which  is  alkaline  to 
litmus  is  acid  to  phenolphthalein,  showing  that  there  are  present  in  such 
media  substances  possessing  an  acid  character  which  litmus  does  not 
detect.  These  substances  are  weak  organic  acids  and  organic  com- 
pounds, theoretically  amphoteric,  but  in  which  an  acid  character  pre- 
dominates. Thus,  a  litre  of  bouillon  becomes,  on  the  addition  of  1  per 
cent,  of  peptone,  more  alkaline  to  litmus,  but  decidedly  more  acid  to 
phenolphthalein;  100  c.c.  of  water  with  1  per  cent,  of  peptone  is  acid 
to  phenolphthalein  to  such  an  extent  that  3.5  c.c.  of  decinormal  NaOH 
is  required  to  neutralize  it.  To  litmus  it  is  alkaline  and  requires  3.4  c.c. 
of  decinormal  HC1.  Two  per  cent,  of  peptone  doubles  the  difference. 
The  same  figures  hold  approximately  true  for  peptone  broth.  We 
should  find  by  growing  the  bacteria  just  what  reaction  we  want  for  any 
variety,  and  then  test  the  fluid  with  phenolphthalein  or  litmus  as  the 
indicator.  With  precisely  similar  ingredients  we  can  then  exactly  re- 
produce at  any  time  in  the  future  the  same  reaction,  but  with  different 
materials  one  would  again  have  to  study  the  reaction. 

Titration  of  Culture  Media.  —  We  must  have  accurately  standard- 
ized normal  and  decinormal  solutions  of  sodium  hydrate  and  hydro- 
chloric acid ;  also  a  0.5  per  cent,  solution  of  phenolphthalein  in  30  per 
cent,  alcohol  and  a  neutral  1  per  cent,  solution  of  Merck's  litmus. 

Care  should  be  taken  to  prevent  the  absorption  of  carbon  dioxide  by 
the  soda  solution,  by  arranging  that  all  air  which  comes  in  contact  with 
the  latter,  either  in  the  stock  bottle  or  in  the  burette,  shall  first  pass 
through  a  strong  solution  of  sodium  or  barium  hydrate.  The  arrange- 
ment of  the  apparatus  is  described  in  any  work  on  chemical  analysis. 
The  medium  is  brought  to  the  desired  volume  with  wrater,  and  boiled 
four  minutes  to  expel  the  carbon  dioxide.  Media  are  commonly  warm  or 
hot  when  measured,  hence  it  must  be  remembered  that  true  volumes 
cannot  be  thus  obtained ;  for  instance,  a  litre  measured  at,  say,  80°  C. 
would  be  only  973  c.c.  if  measured  at  20°  C.,  the  temperature  at  which 
litre  flasks  are  calibrated.  Since  many  media  cannot  be  cooled  to  20°  C. 
because  of  solidification,  as  in  the  case  of  agar  or  gelatin,  it  is  a  better 
plan  when  accuracy  is  important  to  determine  measures  of  volume  by 
weight.  For  this,  place  a  clean,  dry  saucepan,  in  which  the  medium  is 
to  be  prepared,  upon  one  side  of  a  trip  scale,  and  counterbalance  its 
weight  exactly.  The  weight  of  a  litre  of  bouillon,  gelatin,  or  agar 
having  been  determined  once  for  all,  the  necessary  weights  added  to 
the  weight  of  the  pan  will  give  the  amount  which  the  pan  and  its  con- 


54  PRINCIPLES  OF  BACTERIOLOGY 

tents  must  balance  when  the  volume  is  exactly  one  litre.  A  portion  of 
the  medium  brought  to  the  exact  volume  is  then  taken  and  cooled  to 
room  temperature  (20°  C.),  or  to  a  point  a  few  degrees  above  solidifi- 
cation, and  10  c.c.  withdrawn,  placed  in  a  small  beaker,  50  c.c.  of  dis- 
tilled water  and  1  c.c.  of  the  phenolphthalein  solution  added.  If  the 
medium  is  acid  the  -f$  NaOH  solution  is  then  run  in  cautiously  until  a 
pale  but  decided  pink  color  is  obtained.  The  number  of  cubic  centi- 
metres of  the  solution  used,  multiplied  by  ten,  will  give  the  number 
of  cubic  centimetres  of  normal  sodium  hydrate  per  litre  necessary  to 
effect  complete  neutralization.  The  question  as  to  what  is  the  best 
reaction  of  media  for  general  work  is  not  an  easy  one  to  settle,  and 
one  on  which  bacteriologists  differ.  What  is  the  proper  reaction  for  one 
variety  of  bacteria  is  often  far  from  the  best  for  some  other  variety. 
Reactions  are  now  commonly  expressed  by  plus  or  minus  signs,  the 
former  representing  an  acid  and  the  latter  an  alkaline  condition,  the 
number  following  the  sign  representing  the  percentage  of  normal  acid 
or  alkali  present  in  the  medium.  Thus,  +1.5  would  indicate  that  the 
medium  contained  1.5  parts  per  100  or  1.5  percent,  of  free  normal  acid, 

FIG.  23  FIG.  24 


Krlenmeyer  flask.  Pasteur  flask. 

while  — 1.5  would  indicate  that  the  medium  contained  an  equivalent 
quantity  of  free  alkali.  The  committee  of  the  American  Public  Health 
Association  in  1898  adopted  a  medium  whose  litre  was  +1.5  as  the 
best  for  general  work  in  water  examinations.  In  1905  this  was  changed 
to  +1.0  per  cent.  A  medium  whose  reaction  is  +0.5  per  cent,  acid  to 
phenolphthalein  is  still  better  adapted  for  many  bacteria.  It  cannot  be 
too  strongly  impressed  upon  the  reader  that  whatever  the  reaction,  its 
measure  should  be  stated  in  all  descriptions  of  cultural  characters. 
The  litmus  solution  is  added  in  the  same  way  as  that  of  phenolphthalein. 
Storage  of  Media. — The  nutrient  media  are  stored  in  glass  flasks  (Fig. 
23).  From  these,  as  needed,  glass  tubes  are  filled.  When  small  amounts 
of  media  are  taken  frequently  from  flasks,  Pasteur's  flasks  (Fig.  24)  are 
of  great  convenience.  They  consist  of  a  flask  with  a  ground-glass  neck, 
over  which  fits  a  cap.  This  cap  may  or  may  not  terminate,  as  desired, 
in  a  narrow  tube,  which  is  plugged  with  cotton.  The  cap  keeps  the 
edges  of  the  flask  free  from  bacteria  and  prevents  the  cotton  from 


CULTIVATION  OF  BACTERIA  55 

sticking.     A  tumbler  or  a  simple  cap  of  paper  over  the  neck  answers 
much  the  same  purpose. 

Preparation  and  Filling  of  Tubes. — The  cheaper  grades  of  test-tubes 
should  be  avoided.  They  are  thin  and  break  easily,  and  also  frequently 
frost  on  heating  from  the  separation  of  silicic  acid.  The  tubes  of  the 
better  class  can  be  used  after  rinsing  with  hot  water;  cheap  tubes  are 
very  alkaline  and  must  first  be  soaked  in  dilute  hydrochloric  acid. 

CLEANSING  \XD  STERILIZATION  OF  APPARATUS. — In  order  to  study 
bacteria,  both  in  culture  media  and  in  the  living  body,  we  must,  as 
already  stated,  separate  those  developed  from  one  organism  from  all 
others  and  study  them  by  themselves  in  pure  cultures.  In  order  to  do 
this  we  have  to  take  the  greatest  precautions  to  ensure  that  the  materials 
that  we  make  use  of  for  the  growth  of  bacteria,  the  flasks  and  tubes  that 
hold  these  materials,  and  the  instruments  with  which  we  transfer  the 
bacteria  are  sterile.  In  bacteriological  work  sterilization  is  practically 
always  done  by  means  of  dry  and  moist  heat,  for  no  antiseptic  sub- 
stances can  be  allowed  to  remain  in  any  of  the  media  used  for  the 
growth  of  bacteria  or  on  any  of  the  apparatus  which  would  come  in 
contact  with  them,  as  such  substances  wrould  inhibit  the  growth  of  the 
bacteria  which  we  desired  to  study. 

The  platinum  wires  and  loops  used  in  transferring  bacteria  are  ster- 
ilized by  holding  them  for  a  moment  until  red-hot  in  a  gas  or  alcohol 
flame.  They  should  not  be  used  until  time  enough  has  elapsed  for  them 
to  cool  sufficiently  not  to  injure  the  bacteria  touched  by  them.  Knives, 
instruments,  etc.,  are,  after  thorough  cleansing,  placed  in  boiling  1  per 
cent,  soda  solution  for  three  to  five  minutes.  Hypodermic  needles  are 
sterilized  by  boiling  in  soda  solution,  or,  when  this  is  impossible,  they 
are  first  frequently  rinsed  with  boiling  or  with  very  hot  water,  and  then 
filled  with  a  5  per  cent,  carbolic  acid  solu- 
tion for  at  least  thirty  minutes  and  then 
rinsed  again  with  sterile  water.  New  tubes 
and  flasks  sometimes  require  to  be  washed 
in  a  2  per  cent,  solution  of  nitric  acid,  so 
as  to  remove  any  free  alkali  which  may  be 
present.  They  are  finally  thoroughly  rinsed 
in  pure  water.  Old  tubes,  flasks,  and  other 
glassware  are  boiled  for  about  thirty  minutes 
in  a  5  per  cent,  solution  of  washing  soda, 
and  then  thoroughly  rinsed  off  with  water 
until  perfectly  clean.  If  necessary,  any  dirt 
clinging  to  the  insides  of  the  flasks  and  tubes  "^Dry-heat  sterilizer. 
can  be  removed  by  bristle  brushes  or  suit- 
able swabs.  After  the  tubes  and  flasks  have  been  thoroughly  cleaned 
they  are  plugged  loosely  with  ordinary  cotton-batting,  or,  if  that  is  not 
at  hand,  the  more  expensive  absorbent  cotton.  The  tubes  and  flasks 
with  their  cotton  plugs  and  all  other  glassware  are  sterilized  by  dry 
heat  at  150°  C.  for  one  hour  in  the  dry-heat  sterilizer  (Fig.  25). 

The  sterile  tubes  and  flasks  are  filled  with  the  media,  when  small 


56  PRINCIPLES  OF  BACTERIOLOGY 

quantities  are  used,  by  means  of  a  glass  funnel.  The  main  precaution 
to  be  observed  is  not  to  let  the  media  soil  the  neck  of  the  tubes  and  flasks, 
as  this  would  cause  the  fibres  of  the  cotton  plugs  to  adhere  to  the  sides 
of  the  tubes  when  the  media  dried,  and  make  it  difficult  to  remove 
the  plugs  wholly  when  we  wished  to  inoculate  the  contents  of  the 
tubes. 

The  tubes  and  flasks,  plugged  with  sterile  cotton  and  full  of  media, 
are  put  in  the  steam  sterilizer  for  one-half  hour  on  three  consecutive 
days,  or  in  the  autoclave  for  fifteen  minutes  for  two  consecutive  days. 
A  portion  of  the  tubes  containing  nutrient  agar  are  laid  in  a  slanted 
position  before  cooling,  after  the  final  sterilization,  so  that  a  larger  sur- 
face may  be  obtained. 

THE  CULTIVATION  OF  BACTERIA. 

Bacteria  can  seldom  be  identified  by  their  microscopic  and  staining 
characteristics  alone.  We  can  only  study  their  forms,  arrangement,  and 
motility  or  lack  of  motility.  To  go  beyond  this  we  have  to  grow  the 
micro-organism  in  pure  culture  on  the  various  culture  media  and  per- 
haps also  in  animals.  It  is  necessary,  also,  to  have  the  proper  conditions 
as  to  temperature,  moisture,  access  of  oxygen,  etc. 

When  we  make  cultures  from  any  material,  we  are  very  apt  to  find 
that  instead  of  one  variety  of  bacteria  only  there  are  a  number  present. 
If  such  material  is  placed  in  fluid  media  contained  in  test-tubes,  we  find 
that  the  different  varieties  all  grow  together  and  become  hopelessly 
mixed.  When,  on  the  other  hand,  the  bacteria  are  placed  on  solid 
media  they  develop  about  the  spot  where  they  were  inoculated.  If  dif- 
ferent varieties,  however,  are  placed  too  near  together,  they  overgrow 
one  another;  it  is  thus  advisable  to  have  a  greater  surface  of  nutrient 
material  than  is  given  on  the  slanted  surface  of  nutrient  agar  or  blood- 
serum  contained  in  test-tubes.  This  need  is  met  by  pouring  the  media 
while  warm  on  flat,  cool,  glass  plates  or  into  shallow  dishes. 

Technique  of  Making  Plate  Cultures. — In  making  plate  cultures  two 
methods  are  carried  out.  In  the  first  the  material  with  its  con- 
tained bacteria  is  scattered  throughout  the  fluid  before  it  hardens;  in 
the  second  it  is  streaked  over  the  surface  of  the  medium  after  it  has 
solidified.  Nutrient  agar  and  nutrient  gelatin,  the  two  substances  used 
for  plate  cultures,  differ  in  two  essential  points,  which  cause  some  differ- 
ence in  their  uses.  Nutrient  1  per  cent,  agar  melts  near  the  boiling 
point  and  begins  to  thicken  at  about  36°  C.  It  is  not  liquefied  by  bac- 
terial ferments.  Nutrient  10  per  cent,  gelatin  melts  according  to  the 
variety  used,  at  the  low  temperature  of  about  23°  to  27°  C.,  and  solidifies 
at  a  point  slightly  below  that.  It  is  liquefied  by  many  bacterial  ferments. 
When  we  wish  to  inoculate  fluid  nutrient  agar  for  plate  cultures  we  have 
to  take  great  care  that  in  cooling  it  to  a  point  which  will  not  injure  the 
bacteria,  about  41°  C.,  we  do  not  allow  it  to  cool  too  much  and  thus 
solidify  and  prevent  our  pouring  it  into  the  plates.  To  prevent  this, 
when  a  number  of  tubes  are  to  be  inoculated,  they  are  placed  while  still 


CULTIVATION  OF  BACTERIA 


FIG.  26 


hot  in  a  basin  of  water  which  has  been  heated  to  about  45°  C. 
\Yhen  the  temperature  of  the  agar  in  the  tubes,  as  shown  by  a  ther- 
mometer in  one,  has  fallen  to  42°  C.,  the  water,  milk,  iVces,  bacterial  cul- 
ture, or  other  substance  to  be  tested  is  added  to  the  other  tubes  in  what- 
ever quantity  is  thought  to  be  proper  up  to  1  c.c.  A  greater  quantity 
of  fluid  would  dilute  and  cool  the  nutrient  agar  too 
much.  After  inoculation  the  contents  of  the  tubes 
are  thoroughly  shaken  and  poured  out  quickly  into 
round,  flat-bottomed,  glass  Petri  dishes  (Fig.  26), 
the  covers  of  wliich  are  removed  for  the  required 
time  only.  Instead  of  placing  the  substance  in 
the  tube  it  is  often  placed  directly  in  the  Petri 
dish.  The  melted  nutrient  gelatin  or  agar  is  thus  poured  over  it 
and  by  gently  tipping  the  dish  mixed  with  it.  The  bacteria  are  now 
scattered  throughout  the  fluid,  and  as  it  quickly  solidifies  they  are  fixed 

FIG.  27 


Petri  dish. 


Photograph  of  a  large  number  of  colonies  developing  in  a  layer  of  gelatin  contained  in  a  Petri 
dish.  Some  colonies  are  only  pinpoint  in  size ;  some  as  large  as  a  pencil.  The  colonies  here  appear 
in  their  actual  size. 

wherever  they  happen  to  be,  and  thus  as  each  individual  multiplies 
clusters  are  formed  about  it  at  the  spot  where  it  was  fixed  at  the  moment 
of  solidification.  The  number  of  colonies  of  bacteria  (Fig.  27)  thus 
indicate  to  us  roughly  the  number  of  living  bacteria  in  the  quantity 
of  fluid  added  to  the  liquid  agar.  Groups  or  chains  of  bacteria 
which  in  spite  of  shaking  remain  attached  produce  single  colonies. 
Nutrient  gelatin  is  used  exactly  as  agar,  except  that  as  the  average 
product  does  not  congeal  until  cooled  below  22°  C.  we  have  no  fear 
of  its  cooling  too  rapidly.  In  order  not  only  to  count  the  number  of 
colonies  which  develop,  but  also  to  obtain  a  characteristic  'growth,  it  is 
desirable  not  to  have  them  too  near  together. 


58 


PRINCIPLES  OF  BACTERIOLOGY 


Dilution  Methods. — As  it  is  impossible  to  know  the  number  of  bac- 
teria in  any  suspected  fluid,  it  is  usual  to  make  a  set  of  four  different 
plates,  to  each  of  which  a  different  amount  of  material  is  added,  so  that 
some  one  of  the  four  will  have  the  required  number  of  colonies.  The 
dilutions  are  made  in  sterile  distilled  water  or  bouillon.  In  the  first  tube 
we  place  an  amount  which  we  believe  will  surely  contain  sufficient 
and  probably  too  many  bacteria.  To  the  second  tube  we  add  10  per 
cent,  of  the  amount  added  to  the  first,  and  to  the  third  10  per  cent, 
of  the  second,  and  to  the  fourth  10  per  cent,  of  the  third.  Thus,  if  the 
first  contained  60,000  colonies  the  second  would  have  6000  (Fig.  27), 
the  third  600,  and  the  fourth  60.  If,  on  the  other  hand,  the  first  con- 
tained but  60,  the  second  would  have  about  6,  and  the  remaining  two 
would  probably  contain  none  at  all.  When  there  are  many  colonies 
present  the  dishes  are  covered  by  a  glass  plate  (Fig.  29),  ruled  in  larger 
and  smaller  squares,  Wolffhiigers  apparatus.  With  the  eye  or  aided  by 
a  hand  lens  the  colonies  in  a  certain  number  of  squares  are  counted 
and  then  the  number  for  the  whole  contents  estimated. 


FIG.  28 


FIG.  29 


Well-distributed  colonies  on  agar  in 
Petri  dish. 


Wolff  hiigel's  apparatus  for  counting  colonies. 


When  the  material  to  be  tested  is  crowded  with  bacteria  it  is  often 
best  to  make  an  emulsion  of  a  portion  of  it,  and  use  this  rather  than 
the  original  substance  for  making  the  dilutions  to  be  used.  Measured 
quantities  of  the  diluted  material  can  be  transferred  most  accurately 
through  a  sterilized,  long,  glass  pipette  graduated  in  one  one-hundredth 
cubic  centimetres,  or,  more  roughly,  by  a  platinum  loop  of  known  size. 

The  nutrient  agar-agar  is  frequently  used  in  a  different  manner. 
About  8  c.c.  are  poured  into  the  Petri  dish  and  allowed  to  harden. 
The  substance  to  be  tested  bacteriologically,  or  a  dilution  of  it,  is  then 
drawn  across  the  medium  in  a  series  of  parallel  streaks  by  means  of  a 
platinum  loop  lightly  over  its  surface.  Each  successive  streak  is  made 
with  the  same  needle  or  loop  without  replenishing  the  material  to  be 
tested.  Each  streak  will  therefore  leave  less  deposit  of  bacteria  and 
fewer  colonies  will  develop.  While  in  the  former  method  most  of  the 
bacteria  developed  under  the  surface,  here  all  develop  upon  it.  This 


CULTIVATION  OF  BACTERIA  59 

is  an  advantage,  as  many  forms  of  bacteria  develop  more  character- 
istically on  the  surface  than  in  the  midst  of  the  media,  and  it  is  easier 
to  remove  them  free  from  other  bacteria  with  the  platinum  needle. 
Instead  of  streaking  the  material  by  means  of  the  platinum  wire  over 
the  agar,  a  loopful  may  be  deposited  on  the  agar  and  then  smeared  over 
its  surface  by  a  sterile  swab  or  bent  glass  rod.  The  method  of  using 
glass  plates  upon  a  cooling  stage  has  now  been  practically  given  up  for 
the  more  convenient  one  of  Petri  dishes.  In  warm  weather  the  dishes 
may  be  cooled  before  using,  so  as  to  harden  quickly  the  agar  or  gelatin 
that  is  poured  into  them. 

An  old  method,  which  is  still  sometimes  used  to  find  the  number  of 
living  bacteria,  is,  instead  of  pouring  out  the  media  which  has  been 
inoculated,  to  congeal  it  on  the  sides  of  the  test-tubes.  This  is  best 
done  by  laying  the  tube  flat  on  its  side  on  a  cake  of  ice  and  rotating  it. 
Tubes  come  especially  formed  for  this  by  having  a  slight  neck,  which 
prevents  the  media  running  up  to  the  plugged  end  of  the  tube.  This 
method,  Esmarch's,  is  used  only  when  the  Petri  dishes  are  not  obtain- 
able or  cannot  easily  be  transported. 

Study  of  Colonies  in  Plate  Cultures  in  Nutrient  Agar. — The  plates 
should  be  removed  after  twelve  to  twenty-four  hours'  growth  at  blood 
temperature  and  after  one  to  three  days  at  70°  F.  (21°  C.).  The 
special  time  allowed  varies  according  to  the  rapidity  of  the  growth  of 
the  varieties  developing;  thus,  bacteria,  such  as  the  streptococci  and 
influenza  bacilli,  reach  the  characteristic  development  of  their  colonies 
in  from  ten  to  sixteen  hours,  while  others  continue  to  spread  for  several 
days.  If  we  wait  too  long  where  numerous  varieties  of  bacteria  are 
growing  the  colonies  of  heavier  growth  may  cover  up  the  finer  and 
more  delicate  ones.  As  a  rule,  the  younger  colonies  are  more  charac- 
teristic, except  where  the  development  of  pigment  is  sought. 

The  colonies  are  first  examined  with  the  eye  (Fig.  27),  then  with 
magnification  of  about  60  diameters  (Figs.  30  to  35),  and  then  again, 
if  necessary,  at  from  400  to  500  diameters  (Fig.  44).  We  note  every- 
thing we  can  about  them,  such  as  their  size,  surface  elevation,  form, 
internal  structure,  edges,  and  optical  characters.  If  grown  in  gelatin, 
whether  they  have  or  have  not  caused  liquefaction.  The  accompanying 
schematic  representations  from  Lehman  and  Neumann  (Figs.  36  to  43) 
illustrate  some  of  these  points. 

At  the  higher  magnification  we  begin  to  detect  the  individual  bac- 
teria (Fig.  44).  After  studying  the  colonies  we  remove  a  few  of  the 
bacteria  from  one  or  more  of  them  by  touching  them  with  the  tip  of  a 
sterile  platinum  needle  (Fig.  45),  and  thus  transfer  them  to  a  cover- 
glass  for  microscopic  examination,  or  to  new  media  where  they  may 
develop  in  pure  cultures  and  show  their  growth  characteristics. 

Hanging  Block  Cultures.  —  In  order  to  study  the  morphology  and 
manner  of  multiplication  of  bacteria  to  better  advantage  than  in 
the  hanging  drop,  Hill  devised  the  following  procedure:  Melted 
nutrient  agar  is  poured  into  a  Petri  dish  to  a  depth  of  about  one-eighth 
to  one-quarter  of  an  inch.  When  cool  a  block  is  cut  out  about  one- 


60 


PRINCIPLES  OF  BACTERIOLOGY 


quarter  of  an  inch  square.  The  block  is  placed  under  surface  down  on 
a  slide  and  protected  from  dust.  A  suspension  of  the  growth  to  be 
examined  is  then  made  in  sterile  bouillon  and  spread  over  the  upper 


FIG.  30 


FIG.  31 


FIG.  30.— Irregular  fringed  colony  (B.  malignant  cedema).    (From  Kolle  and  Wasserman.) 
FIG.  31.— Round  surface  colony  (colon  bacilli  grown  in  stiff  gelatin). 


FIG.  32 


FIG.  33 


FIG.  32.— Colony  of  typhoid  in  rather  stiff  gelatin. 

FIG.  33.— Colonies  of  typhoid  and  colon  bacilli  in  rather  soft  gelatin. 


FIG.  34 


FIG.  35 


FIG.  34.— Colony  of  colon  bacilli  grown  in  soft  gelatin. 

FIG.  35.— One  irregular  colony  of  colon  and  two  of  typhoid  bacilli  in  soft  gelatin.    (Figs.  31-35  from 
photographs  by  Dunham.) 

surface  of  the  block.  The  slide  and  block  are  then  put  in  the  incubator 
for  ten  minutes  to  dry  slightly.  A  clean  cover-slip  is  now  placed  on 
the  agar  block  in  such  a  way  as  to  avoid  large  air  bubbles.  The  slide 


CULTIVATION  OF  BACTK^l.\ 


61 


is  then  removed.     With  the  aid  of  a  platinum  loop  a  drop  or  two  of 
melted  agar  is  run  along  each  side  of  the  block  to  fill  any  angles  between 


FIG.  36 


FIG.  37 


FIG.  36.— Moist  raised  colonies  with  no  visible  structure,  looking  like  a  drop  of  water. 
FIG.  37.— Deep  colonies,  usually  either  light  brown,  gray  or  yellow  in  color,  opaque,  with  little 
marking.    (Figs.  36-43  from  Lehman  and  Neumann.) 


FIG.  38 


FIG.  39 


FIG.  40 


FIG.  38.— The  colonies  very  finely  granular,  with  or  w  ithout  twisted  threads  at  borders. 
FIG.  39.— Colonies  opaque  in  centre  with  lighter  borders.    The  margin  is  coarsely  granular. 
FIG.  40.— Colony  in  gelatin.    The  centre  is  coarsely  granular  in  partly  fluid  gelatin.    The  borders, 
are  formed  of  wavy  bands  of  threads. 


FIG.  41 


FIG.  42 


FIG.  43 


.  •;•  - 


FIG.  41.— Colonies  circular  in  form,  composed  of  radiating  threads. 

FIG.  42.— Colonies  with  opaque  centres,  with  a  thin  border  fringe. 

FIG.  43.— Colony  showing  a  network  of  threads  which  is  thicker  in  centre. 

it  and  the  cover-glass.     After  drying  in  the  incubator  for  five  minutes 
it  is  placed  over  a  hollow  slide  and  sealed  with  paraffin. 

In  using  nutrient  gelatin  one  must  always  remember  not  to  allow  it 
to  stay  where  the  temperature  is  over  20°  C.,  for  if  that  happens  the 


62 


PRINCIPLES  OF  BACTERIOLOGY 
FIG.  44  FIG.  45 

fH)      Ct 


H 


Two  surface  colonies  of  diphtheria  bacilli  upon  agar. 
X  500  diameters. 

FIG.  46 


Platinum  needle,  loop, 
and  spade. 

media  as  a  rule  will  melt; 
nor  must  the  liquefying  col- 
onies be  allowed  to  grow  for 
too  long  a  time,  or  the  entire 
media  will  become  fluid. 

Pure  Cultures.  —  If  we 
transfer  without  contamina- 
tion bacteria  from  a  colony 
formed  from  a  single  organ- 
ism to  new  media,  and  these 
grow,  we  have  what  we  call 
a  pure  culture  of  that  variety. 
When  these  are  transferred  to  the  solid  media  we  call  the  growth  which 
takes  place  from  smearing  the  bacteria  over  the  surface  a  surface  or 
smear  culture,  and  that  formed  in  the  depth  of  the  media  by  plunging 
the  needle  carrying  the  bacteria  into  it  a  stab  culture  (Figs.  46  and  47). 
In  transferring  bacteria  from  one  tube  to  another  we  slant  the  tubes 
so  that  no  dust  may  fall  within  and  contaminate  with  other  bacteria 
the  special  variety  we  wish  to  transplant.  The  greatest  care  must  be 
taken  that  the  sterilized  platinum  needle  used  to  transfer  the  bacteria 
is  not  infected  by  touching  any  non-sterile  matter.  The  upper  rim 
of  culture  tubes  should  be  passed  through  the  flame  so  as  to  destroy 
any  bacteria  resting  there.  Even  with  our  utmost  care  bacteria  will 
from  time  to  time  pass  from  the  air  or  edges  of  our  tubes  into  the  culture 


Stab  cultures  of  three  cholera  spirilla  in  gelatin,  show- 
ing in  upper  portion  of  growth  considerable  liquefaction 
of  nutrient  gelatin. 


CULTIVATION  OF  BACTERIA 


63 


media,  and  thus  possibility  of  contamination  must  always  be  kept  in 
mind.  When  it  occurs  upon  solid  media  we,  as  a  rule,  easily  detect  it, 
for  we  notice  the  growth  at  some  point  of  bacteria  of  different  colony 
characteristics;  but  in  fluid  media,  on  account  of  the  complete  mingling 
of  the  bacteria,  we  are  not  so  apt  to  notice  the  additional  growth. 

THE  STUDY  OF  PURE  CULTURES  ix  TUBED  MEDIA. — A  few  points 
of  the  many  which  should  be  observed  are  the  following: 

Gelatin  stab  cultures. 

A.  Xon-liquefying. 

Line  of  puncture. 

Filiform,  uniform  growth,  without  special  characters. 
Beaded,  consisting  of  loosely  placed,  disjointed  colonies. 
Arborescent,  branched,  or  tree-like. 
Some  of  these  points  are  illustrated  in  Fig.  47,  sketched  by  Chester 

B.  Liquefying. 

Crateriform,  a  saucer-shaped  liquefaction  of  the  gelatin. 
Saccate,  shape  of  an  elongated  sac,  tubular  (Fig.  46). 
Statiform,  liquefaction  extending  to  the  walls  of  the  tube. 


FIG.  47 


Showing  characters  of  gelatin  stab  cultures  :  .4.  Characters  of  surface  elevation  :  1,  flat ;  2,  raised  ; 
3,  convex  ;  4,  pulvinate ;  5,  capitate  ;  6,  umbilicate  ;  7,  umbonate.  B.  Characters  of  growth  in  depth  ; 
1,  filiform  ;  2,  beaded ;  3,  tuberculate-ecinulate ;  4,  arborescent ;  5,  villous.  (From  Chester.) 


Nutrient  agar  tube  cultures  give  fewer  points  for  observation,  but 
should  be  studied  in  the  same  way.  The  agar  in  the  tubes  is  usually 
slanted  and  the  culture  growth  is  not  only  in  the  stab,  but  along  the 
streaked  surface.  The  characteristics  of  each  should  be  noticed. 


64 


PRINCIPLES  OF  BACTERIOLOGY 


Apparatus  for  Obtaining  a  Suitable  Temperature  for  the  Growth  of  Bac- 
teria. INCUBATORS. — In  order  to  have  a  constant  and  proper  temper- 
ature for  the  growth  of  bacteria,  forms  of  apparatus  called  incubators 
have  been  devised  (Fig.  48).  These  consist,  in  their  simplest  form,  of 
an  inner  air  chamber  surrounded  by  a  double  copper  wall  containing 
water.  The  apparatus  externally  is  lined  with  asbestos,  to  prevent  radia- 
tion. It  is  supplied  with  doors  and  with  openings  for  thermometers 
and  a  thermoregulator.  The  thermoregulators  are  of  various  kinds; 
those  in'  most  use  depend  upon  the  expansion  or  contraction  of  the 
fluid  in  the  bulb  A  (Fig.  49),  which  rests  within  the  water-jacket,  to 
lessen  or  increase  the  space  between  the  surface  of  the  mercury  B 
and  the  inner  tube  D,  thus  allowing  of  the  passage  of  a  greater  or  less 


FIG.  48 


FIG.  49 


Small  incubator. 


Thermoregulator. 


quantitity  of  gas  to  the  burner  through  the  tube  D.  Other,  forms 
depend  upon  the  contraction  or  expansion  of  metal,  or  the  use  of  the 
electric  current  to  control  the  flow  of  the  gas. 

The  temperature  in  the  air  chamber  is  kept  above  that  of  the  sur- 
rounding air  by  means  of  a  gas  flame  regulated  as  above  described, 
or,  when  that  cannot  be  obtained,  a  lamp. 

The  temperature  is  reduced  by  passing  a  stream  of  cool  water  through 
the  water  chamber,  which  is  itself  regulated.  When  very  accurate 
investigations  are  to  be  made  a  gas-pressure  regulator  is  added  to  the 
thermoregulator.  Incubators  are  also  both  warmed  and  regulated 
by  electricity. 

In  emergencies  a  culture  may  be  developed  at  the  blood  tempera- 
ture by  placing  it  in  water  contained  in  a  small  vessel,  which  itself 
is  contained  in  a  larger  vessel,  also  filled.  By  adding  a  little  hot  water 


CCLTIVATIOX  nr  BACTERIA  65 

from  time  to  time  to  the  outer  vessel  the  temperature  can  readily  !>e 
kept  between  34°  and  38°  C.,  which  is  sufficiently  uniform  for  bacteria 
such  as  the  diphtheria  bacilli  to  grow. 

As  a  temporary  expedient  during  the  night,  when  haste  is  necessary, 
it  is  possible,  when  the  culture  medium  is  solid  and  within  a  strong 
glass  tube  or  metal  case,  to  make  use  of  the  body  heat  by  putting  it 
under  the  clothing  next  to  the  body  or  sleeping  upon  it.  Naturally, 
this  should  only  be  done  when  other  means  fail.  Several  times,  when 
in  the  country,  this  method  has  enabled  the  writer  to  obtain  a  growth 
of  diphtheria  bacilli  over  night,  and  thus  get  important  information, 
when  otherwise  it  would  have  been  impossible. 

Methods  for  Obtaining  Anaerobic  Conditions  for  Bacteria. — Pasteur 
excluded  the  oxygen  by  pouring  a  layer  of  oil  on  the  culture  fluid.  A 
simple  device  is  that  of  Koch,  who  placed  a  thin  strip  of  sterile  mica 
upon  the  still  fluid  agar  or  gelatin  in  the  Petri  dish,  which  had  already 
been  inoculated.  After  the  solidification  of  the  media  the  portion 
under  the  mica  is  excluded  from  the  air  and  anaerobic  growth  can 
develop. 

A  second  simple  method  (Liborius)  is  to  fill  the  tubes  with  media 
fuller  than  usual  and  to  inoculate  the  bacteria  deep  down  to  near 
the  bottom  of  the  tubes  while  the  media  are  still  semisolid.  An 
anaerobic  growth  will  take  place  in  the  lower  part  of  the  tube.  In  a 
similar  way  the  closed  arm  of  the  fermentation  tube  will  suffice  for 
anaerobic  growth,  if  the  opening  connecting  it  with  the  open  bulb  is 
quite  small.  Wright  devised  the  following  procedure:  A  short  glass 
tube  with  constricted  ends  is  used.  Each  end  has  a  piece  of  rubber 
tubing  attached.  One  of  these  is  connected  with  a  glass  tube,  which 
projects  through  the  cotton  plug  of  the  test-tube.  The  test-tube  con- 
tains bouillon.  The  whole  is  sterilized  and  then  the  test-tube  inocu- 
lated. The  bouillon  is  then  drawn  up  into  the  constricted  tube,  which 
is  sealed  by  simply  pushing  down  the  tube  so  that  both  rubber  ends 
are  sealed  by  being  bent  on  themselves.  When  spores  are  present,  a 
simple  method  suggested,  I  believe,  by  McFarland,  can  be  success- 
fully employed.  Vessels  plugged  with  stoppers  perforated  by  glass 
tubes  drawn  to  a  point  are  filled  to  such  a  height  that  when  the  fluid 
is  heated  to  80°  C.  it  will  just  fill  them.  They  are  inoculated  when 
the  bouillon  is  at  about  60°  C.,  heated  to  80°  C.,  and  then  sealed  by 
closing  the  tube's  point  by  means  of  a  flame.  After  inoculating  and 
heating,  instead  of  sealing  the  glass  tube  a  sterile  rubber  cork  can  be 
inserted. 

If  much  fermentation  is  expected,  the  cork  should  be  clamped  or  tied 
to  the  bottle,  so  that  it  will  not  blow  out.  One  advantage  of  this  method 
is  that  any  contaminating  organisms  which  have  no  spores  will  be 
killed. 

When  sealed  the  bottles  should  be  cooled  and  then  placed  in  the 
incubator. 

A  very  convenient  modification  of  Pasteur's  method  for  the  growth 
of  bacteria  in  fluid  media  is  to  cover  the  fluid  with  albolene  or  paraffin. 

5 


66 


PRINCIPLES  OF  BACTERIOLOGY 


In  boiling  all  the  oxygen  is  driven  out.  We  prepare  all  our  tetanus 
toxins  in  this  way:  Litre  flasks  are  filled  to  near  the  neck  with  bouillon. 
This  is  covered  with  a  one-half  inch  layer  of  albolene  or  paraffin.  The 


FIG.  50 


Jar  for  anaerobic  cultures. 

bouillon  after  boiling  is  quickly  cooled  by  setting  the 
flask  containing  it  in  a  shallow  layer  of  cool  water, 
so  as  to  lower  the  temperature  of  the  lower  portion 
of  the  bouillon  to  40°  C.  or  under,  while  leaving  the 
paraffin  on  the  surface  still  fluid.  While  in  this  con- 
dition it  is  inoculated  with  the  tetanus  culture.  Bits 
of  tissue  suspected  to  contain  tetanus  bacilli  are 
dropped  into  smaller  flasks  filled  and  prepared  in 
the  same  way. 

DISPLACEMENT  OF  AIR. — In  the  more  complicated 
methods  the  plates  or  tubes  are  placed  in  jars  of  a 
type  devised  by  Novy  (Fig.  50),  in  which  the  oxygen 
is  displaced  by  a  stream  of  hydrogen  developed  by 
the  Kipp  apparatus,  through  the  action  of  pure  granu- 
lated zinc  and  a  25  per  cent,  solution  of  pure  sul- 
phuric acid.  When  all  the  oxygen  has  been  displaced 
the  jars  are  sealed  by  rotating  the  stopper. 

ABSORPTION  OF  OXYGEN. — In  another  method  the 
oxygen  is  extracted  by  a  mixture  of  pyrogallic  acid 
and  caustic  potash.  To  each  100  c.c.  of  air  space  in 
the  jar  1  gram  of  pyrogallic  acid  and  10  c.c.  of  6 
per  cent,  solution  of  potassium  hydroxide  are  added 
and  the  jars  immediately  sealed.  A  very  simple  pro- 
cedure has  been  described  by  Wilson.  In  a  large  test- 
tube  a  small  piece  of  solid  caustic  potash  is  placed 
and  over  this  powdered  pyrogallic  acid  is  poured. 
A  smaller  culture  tube  with  the  desired  medium  is 
inoculated.  Water  is  now  added  to  the  large  test- 
tube,  which  works  its  way  slowly  through  the  pyro- 


FlG.  51 


Buchner's  anaerobic 
tube.  The  fluid  consists 
of  pyrogallic  acid  dis- 
solved in  10  per  cent, 
soda  solution.  By  Wil- 
son's method  the  tubes 
are  charged  with  pieces 
of  caustic  potash  cover- 
ed with  pyrogallic  acid. 


CULTIVATION  OF  BACTERIA  67 

gallic  acid.  The  small  tube  is  quickly  inserted  and  the  whole  sealed 
by  water  or  a  rubber  cork  (Fig.  51).  Solid  culture  media  in  test-tubes 
can  be  inverted  over  the  acid  soda  mixture,  which  is  then  covered 
by  a  layer  of  albolene  to  prevent  the  absorption  of  oxygen  from  the 
air.  The  displacement  method  is  often  used  along  with  that  of 
absorption. 

ASSOCIATED  WITH  AEROBIC  BACTERIA. — Anaerobic  bacteria  mixed 
with  aerobic  bacteria  will  grow  in  the  apparent  presence  of  oxygen, 
the  aerobic  bacteria  robbing  the  media  of  it.  Thus,  tetanus  and 
diphtheria  grow  together  in  an  open  flask  of  bouillon. 

METHOD  FOR  ADAPTING  BACTERIA  TO  ANIMAL  FLUIDS. — The  placing 
of  cultures  in  collodion  sacs  in  the  abdomens  of  animals  has  been  used 
extensively  by  the  Pasteur  school  for  exalting  the  virulence  of  bacteria 
or  trying  to  adapt  them  to  species  of  animals  differing  from  the  one 
from  which  they  were  isolated. 

The  underlying  idea  is  to  grow  the  organisms  in  the  peritoneal  cavity 
of  an  animal  under  such  conditions  that  the  waste  products  of  the 
germs  will  be  removed,  an  abundant  supply  of  nutrient  material  fur- 
nished, and  the  germs  themselves  protected  from  the  action  of  the 
phagocytes.  The  hermetically  sealed  collodion  sacs  answer  this  pur- 
pose. The  collodion  used  is  the  U.  S.  Pharmacopoeia  solution,  which 
by  exposure  to  the  air  has  been  concentrated  one-third. 

The  sealed  inoculated  sacs  are  to  be  inserted  into  the  peritoneal 
cavity  with  every  possible  precaution  for  asepsis.  The  sacs  are  left 
in  place  for  days  or  months,  as  the  experiment  requires. 


CHAPTEE  VI. 

MICROSCOPIC  METHODS. 

THE  PREPARATION,  STAINING,  AND  MICROSCOPIC  EXAMINATION 

OF  BACTERIA. 

THE  direct  microscopic  examination  of  suspected  substances  for 
bacteria  can  be  made  either  with  or  without  staining.  Unstained, 
the  bacteria  are  examined  in  a  hanging  drop  or  on  transparent  media, 
under  daylight,  or,  better,  artificial  light  to  note  their  motility,  their  size 
and  form,  and  their  general  arrangement;  but  for  more  exact  study 
of  their  appearance  they  can  be  so  much  better  observed  when 
stained  that  this  step  is  always  advisable. 

Elimination  of  Foreign  Bacteria  from  Preparations. — Since  bacteria 
are  present  in  the  air,  in  dust,  in  tap  water,  on  our  bodies,  clothes,  and 
all  surrounding  objects,  it  follows  that  when  we  begin  to  examine  sub- 
stances for  bacteria  the  first  requisite  is  that  the  materials  we  use, 
such  as  staining  fluid,  cover-glasses,  etc.,  should  be  practically  free 
from  bacteria,  both  living  and  dead,  otherwise  we  may  not  be  able  to 
tell  whether  those  we  detect  belonged  originally  in  the  substances 
examined  or  only  in  the  materials  we  have  used  in  the  investigation. 

Film  Preparation. — A  cover-glass  or  slide  preparation  is  made  as 
follows:  A  very  small  amount  of  the  blood,  pus,  discharges  from 
mucous  membranes,  cultures  from  fluid  media,  or  other  material  to 
be  examined  is  removed  by  means  of  a  sterile  swab  or  platinum  loop 
and  smeared  undiluted  in  an  even,  thin  film  over  a  perfectly  clean,1 
thin  cover-glass  or  slide.  From  cultures  on  solid  media,  however, 
on  account  of  the  abundance  of  bacteria  in  the  material,  a  little  of  the 
growth  is  diluted  by  adding  it  to  a  tiny  drop  of  clean  distilled  water 
which  has  been  previously  placed  on  the  glass.  The  amount  of  dilution 
is  learned  after  a  few  trials.  It  is  best  to  add  to  the  drop  just  enough 
of  the  culture  to  make  a  perceptible  cloudiness.  The  mixture  is  then 
smeared  thinly  and  uniformly  over  the  glass.  When  blood  or  pus  is 
to  be  studied  it  is  well  to  put  a  small  drop  on  a  slide  or  cover-glass 
and  then  immediately  to  place  on  top  of  this  another.  The  fluid  will 
spread  between  the  two,  and  when  they  are  drawn  apart  a  fairly  even 

1  To  render  new  cover-slips  clean  and  free  from  grease,  place  them  in  strong  nitric  acid  for  a  few 
hours,  then  rinse  them  in  water,  then  in  alcohol,  then  in  ether.  Place  them  finally  for  keeping 
in  alcohol,  to  which  a  little  ammonia  has  been  added.  When  used  wipe  with  soft,  clean  linen  or 
cotton  cloth.  If  old  cover-slips  are  used,  boil  first  in  soda  solution.  Another  procedure  is  to  soak 
the  cover-glasses  first  in  alcohol,  then  wipe  with  soft  linen,  then  place  in  a  Petri  dish,  and  heat  in 
the  dry  sterilizer  for  one  hour  at  200°  C.  A  cover-glass  is  not  clean  when  a  drop  of  water  spread  over 
it  does  not  remain  even,  but  gathers  in  droplets. 


MICROSCOPIC  METHODS  69 

smear  will  he  left  on  them.  If  it  is  desired  to  preserve  the  blood  cells 
intact  the  films  are  placed  in  a  saturated  solution  of  corrosive  sublimate 
for  two  to  three  minutes,  and  then  washed  in  running  water,  or  instead 
of  sublimate  exposed  to  the  vapor  of  formalin. 

When  milk  films  are  made  they  are,  after  fixation,  cleared  of  fat  by 
means  of  ether.  From  whatever  source  derived  the  film  is  allowed  to 
dry  thoroughly  at  the  usual  air  temperature,  and  then,  in  order  to  fix 
the  film  with  its  contained  bacteria  to  the  glass,  the  latter  is  grasped  in 
any  one  of  the  several  kinds  of  forceps  commonly  used,  and  is  passed 
three  times  by  a  rather  slow  movement  through  the  Bunsen  or  alcohol 
flame.  Instead  of  this  method  the  film  may  be  fixed  to  the  glass  by 
placing  it  in  absolute  alcohol  for  a  few  minutes.  The  smear  thus  pre- 
pared is  usually  stained  either  by  the  simple  addition  of  a  solution  of 
an  aniline  dye,  for  from  one  to  five  minutes,  or  by  one  of  the  more 
complicated  special  stains  described  later.  When  the  stain  is  to  be 
hastened  or  made  more  intense  the  dye  is  used  warm.  For  ordinary 
staining  the  bacteria  are  simply  covered  completely  by  the  cold  staining 
fluid. 

The  cover-glass  or  slide,  with  the  charged  side  uppermost,  may 
either  rest  on  the  table  or  be  held  by  some  modification  of  Cornet's 
forceps.  When  the  solution  is  to  be  wanned  the  cover-glass  may  be 
floated,  smeared  side  down,  upon  the  fluid  contained  in  a  porcelain 
dish  resting  on  a  wire  mat,  supported  on  a  stand,  or  it  may  be  held  in 
the  Cornet  forceps.  If  a  slide  is  used  it  is  simply  inserted  in  the  fluid 
or  covered  by  it.  The  fluid  in  both  the  dish  and  on  the  glass  should 
be  carefully  warmed,  so  as  to  steam  without  actually  boiling.  The 
glass  should  be  kept  completely  covered  with  fluid.  The  bacteria 
having  now  been  stained,  the  cover-glass  or  slide  is  grasped  in  the 
forceps  and  thoroughly  but  gently  washed  in  clean  water  and  then 
dried,  first  between  layers  of  filter-paper  and  then  in  the  air  or  high 
over  a  flame.  A  drop  of  balsam  or  water  is  now  placed  on  a  glass  slide 
and  the  cover-glass  placed  upon  it  with  the  bacterial  side  down.  The 
cover-glass  or  slide  preparation  is  now  ready  for  microscopic  exami- 
nation. 

Staining  of  Bacteria. — The  protoplasm  of  bacteria  reacts  to  stains 
much  as  nuclear  chromatin,  though  sometimes  more  and  sometimes 
less  actively. 

The  best  stains  are  the  basic  aniline  dyes,  which  are  compounds 
derived  from  the  coal-tar  product  aniline  (C6H5XH2). 

AXILIXE  DYES. — The  aniline  dyes  which  are  employed  for  staining 
purposes  are  divided  into  two  groups  according  as  the  staining  action 
depends  on  the  basic  or  the  acid  portion  of  the  molecule.  The  former 
contains  amido  groups  and  are  spoken  of  as  nuclear  stains,  since  they 
color  the  nuclear  chromatin  of  both  cells  and  bacteria.  The  latter 
contain  hydroxyl  groups  and  do  not  stain  bacteria,  but  are  used  chiefly 
for  contrast  coloring.  The  basic  dyes  are  usually  employed  as  salts 
of  hydrochloric  acid,  while  the  acid  dyes  occur  as  sodium  or  potassium 
salts. 


70  PRINCIPLES  OF  BACTERIOLOGY 

The  following  are  the  most  commonly  used  basic  aniline  stains: 

Violet  stains — methyl  violet,  gentian  violet,  crystal  violet. 

Blue  stains — methylene  blue,  thionin  blue. 

Red  stains — basic  fuchsin,  safranin. 

Brown  stain — Bismarck  brown. 

Green  stain — methyl  green. 

Of  the  above  stains  the  violet  and  red  stains  are  the  most  intense 
in  action.  It  is  thus  easy  to  overstain  a  specimen  with  them.  Of  the 
blue,  methylene-blue  probably  gives  the  best  differentiation  of  struc- 
ture and  it  is  difficult  to  overstain  with  it. 

These  dyes  are  all  more  or  less  crystalline  powders,  and  while  some 
are  definite  chemical  compounds,  others  are  mixtures.  For  this  reason 
various  brands  are  met  with  on  the  market  and  the  exact  duplication 
of  stains  is  not  always  possible.  Dyes  should  be  obtained  from  reliable 
houses  only;  most  bacteriologists  obtain  them  from  Griibler,  of  Leipzig. 
It  is  advisable  to  keep  on  hand  not  only  the  important  dyes,  but  also 
stock  solutions  from  which  the  staining  solutions  are  made.  The  stock 
saturated  alcoholic  solutions  are  made  by  pouring  into  a  bottle  enough 
of  the  dye  in  substance  to  fill  them  to  about  one-quarter  of  their  capacity. 
The  bottle  should  then  be  filled  with  alcohol,  tightly  corked,  well  shaken, 
and  allowed  to  stand  for  twenty-four  hours.  If  at  the  end  of  this  time 
all  the  staining  material  has  been  dissolved,  more  should  be  added, 
the  bottle  being  again  shaken  and  allowed  to  stand  for  another  twenty- 
four  hours.  This  must  be  repeated  until  a  permanent  sediment  of 
undissolved  coloring  matter  is  seen  upon  the  bottom  of  the  bottle.  This 
will  then  be  labelled  "saturated  alcoholic  solution,"  of  whatever  dye 
has  been  employed.  The  alcoholic  solutions  are  not  themselves  em- 
ployed for  staining  purposes.  The  solution  for  use  is  made  by  filling 
a  small  bottle  three-fourths  with  distilled  water,  and  then  adding  the 
concentrated  alcoholic  solution  of  the  dye,  little  by  little,  until  one  can 
just  see  through  the  solution.  Care  must  be  taken  that  the  color  does 
not  become  too  dense;  usually  about  one  part  to  ten  is  sufficient.  Small 
wooden  cases  come  prepared  for  holding  about  one-half  dozen  bottles 
of  the  staining  solutions.  This  number  will  answer  for  all  purposes. 

General  Observations  on  the  Principles  of  Staining  Bacteria. 

Microchemical  Reaction  and  Staining  of  the  Cell  Body. — Of  special 
importance  in  this  regard  is  the  resistance  which  bacteria  possess  to 
diluted  alkalies.  Inasmuch  as  the  majority  of  animal  tissues  are  dis- 
solved when  treated  with  alkalies,  this  method  has  been  adopted  for 
rendering  visible  unstained  bacteria  in  tissues.  As  a  rule,  bacteria 
are  stained  yellowish  with  iodine  solution,  a  few  only  in  consequence 
of  their  starchy  constituents  being  stained  blue. 

Bacteria  may  be  stained  with  various  dyes  of  very  different  chemical 
composition,  such  as  hsematoxylin  and  certain  plant  dyes,  etc.,  but 
most  of  these  are  of  little  practical  value  as  compared  with  the  basic 
aniline  colors.  R.  Koch  was  the  first  to  recognize  the  affinity  of  bacteria 


MICROSCOPIC  METHODS  71 

for  these  dyes  and  to  note  their  importance  as  a  means  of  differentiating 
micro-organisins  from  other  corpuscular  elements. 

The  staining  of  bacteria  is  not  to  be  considered  simply  as  a  mechani- 
cal saturation  of  the  cell  body  with  the  dye,  in  which  the  latter  is  dis- 
solved in  the  plasma.  It  is  rather  a  chemical  combination  between  the 
dye  substance  and  the  plasma.  This  union,  however,  is  apparently 
an  unstable  one  and  easily  broken  up.  Unna  believes  that  the  basic 
aniline  dyes,  from  their  chemical  composition,  are  not  really  bases  but 
neutral  salts — e.g.,  fuchsin  equals  rosaniline  chloride;  they  are  called 
basic  only  because  the  staining  components  (as  the  rosaniline)  are  of  a 
basic  nature.  The  staining  process  is,  therefore,  not  to  be  looked  upon 
as  if  the  dye  substance  separated  into  its  component  parts  and  only  the 
staining  ingredient  attacked  the  cell  body,  because  the  tissues  for  which 
these  "basic  aniline  dyes"  have  special  affinity  are  themselves  basic. 
On  the  contrary,  the  dyestuff  unites  as  a  whole  with  the  plasma,  form- 
ing, as  it  were,  a  double  salt  or  unstable  compound  between  the  two. 

The  dependence  of  the  staining  process  upon  the  solvent  condition 
of  the  dye  is  shown  in  the  following  observations : 

1.  Entirely  water  free,  pure  alcoholic  dye  solutions  do  not  stain. 

2.  Absolute  alcohol  does  not  decolorize  bacteria,  while  diluted  alcohol 
is  an  active  decolorizing  agent.    The  compound  of  dye  substance  and 
plasma  is  therefore  insoluble  in  pure  alcohol. 

3.  The  more  completely  a  dye  is  dissolved  the  weaker  is  its  staining 
power.     For  this  reason  pure  alcoholic  solutions  are  inactive;  and  the 
so-called  weak  dye  solutions  to  which  strong  dye  solvents  have  been 
added  are  limited  in  their  action  on  certain  bacteria  in  which  the  dye 
substance  is  closely  united.     This  is  the  principle  of  Neisser's  stain  for 
diphtheria  bacilli — viz.,  acetic  acid  methylene-blue  solution. 

On  the  other  hand,  the  addition  of  alkalies  to  the  dye  mixture  ren- 
ders the  solvent  action  less  complete,  leading  to  slight  clouding,  and 
finally,  if  large  amounts  of  alkali  are  added,  to  precipitation  of  the 
dye  substance.  Solutions  thus  treated  possess  an  intense  staining 
power.  According  to  Michaels,  however,  in  Loeffler's  methylene- 
blue  solution  the  role  of  the  alkali  is  purely  of  a  chemical  nature,  by 
which  it  converts  the  methylene  blue  into  methylene  azure  (azure  II). 

The  dependence  of  the  staining  process  upon  the  nature  of  the  bac- 
teria is  exhibited  in  the  following  facts: 

There  are  among  bacteria  some  which  are  easily  stained  and  others 
which  are  only  stained  with  difficulty.  To  the  latter  belong,  for  ex- 
ample, the  tubercle  bacillus  and  lepra  bacillus,  also  spores  and  flagella. 
The  difference  between  them  is  that  the  easily  stained  objects  require 
but  a  minimum  of  time  to  be  immersed  in  a  watery  solution,  while 
the  others  must  be  stained  by  special  dyes  or  with  the  aid  of  outside 
influences  (heat  and  mordants,  etc.).  The  difficultly  stained  objects 
are  at  the  same  time  not  easily  decolorized.  The  explanation  of  the 
resistance  which  these  bacteria  show  to  staining  as  well  as  to  decolorizing 
agents  is  to  be  sought  in  two  ways:  either  on  the  assumption  that  they 
possess  a  difficultly  permeable  or  resisting  envelope,  or  that  they  have 


72  PRINCIPLES  OF  BACTERIOLOGY 

a  special  chemical  constitution.  The  latter  hypothesis  holds  good  only, 
if  at  all,  in  regard  to  flagella  and  spores;  while  the  assumption  of  the 
resisting  envelope  has  reference  more  particularly  to  the  tubercle  bacil- 
lus, and  is  probably  correct.  The  presence  of  fatty  and  waxy  bodies 
in  the  envelope  of  these  micro-organisms  is  capable  of  demonstration. 
Moreover,  after  extraction  of  these  bodies  by  ether  the  tubercle  bacillus 
loses  its  power  of  resisting  acids,  which  peculiar  resistance  can  also  be 
artificially  produced  in  other  bacteria  having  normally  no  such  resist- 
ing power.  In  many  instances,  doubtless,  both  of  these  causes,  viz., 
resistant  envelope  and  chemically  different  constitution,  work  together 
to  produce  the  above-mentioned  results. 

Individual  differences  in  acid  resistance  among  the  difficultly  stained 
bacteria  have  been  observed  in  tubercle  bacilli;  according  to  Ziehl  and 
Ehrlich  those  having  less  individual  resistance  are  probably  the  younger 
members.  Individual  differences  in  staining,  in  the  easily  stained  bac- 
teria, have  also  been  noticed;  for  example,  cholera  vibrios  and  allied 
species  are  best  stained  with  fuchsin,  not  so  well  with  methylene  blue,  etc. 

The  relation  between  staining  and  degeneration  of  bacteria  is  a 
complicated  question.  Decrease  of  staining  power  takes  place  during 
degeneration  of  the  bacterial  cell,  but  it  is  often  difficult  to  determine 
the  exact  moment  when  this  loss  of  power  occurs.  Degenerated  forms 
of  the  cholera  bacillus  from  the  abdominal  cavity  of  guinea-pigs  thus 
soon  lose  their  power  of  staining  in  mcthylene-blue  solution,  but  stain 
well  in  diluted  carbol  fuchsin.  Moreover,  bacteria  killed  by  drying  and 
moderate  heating,  as  in  the  preparation  of  films,  retain  their  power  of 
staining.  Kitasato  found  dead  tubercle  bacilli  in  sputum  which  took 
on  normal  staining.  Bacteria  killed  by  chloroform,  formalin,  etc., 
still  retain  their  staining  properties  intact. 

Elective  staining  properties,  whereby  certain  species  of  bacteria  are 
exclusively  or  rapidly  and  intensely  stained  by  certain  dyes,  have  repeat- 
edly been  observed.  Of  the  greatest  practical  importance  in  this  respect 
is  the  Gram  stain  used  for  the  differential  diagnosis  of  many  species  of 
bacteria;  although  a  distinct  classification  of  bacteria  into  those 
which  are  stained  and  those  which  are  not  stained  by  Gram's  solution 
has  been  shown  to  be  impracticable.  There  are  some  bacteria,  how- 
ever, which  act  uniformly  toward  Gram  under  all  conditions;  as,  for 
example,  the  anthrax  bacillus  and  the  pyogenic  cocci  are  always  posi- 
tive, the  cholera  and  plague  bacilli  and  gonococci  are  always  negative 
to  Gram.  Other  species  again  are  at  one  time  stained  and  at  another 
decolorized  by  Gram;  thus  pyocyaneus  is  stained  only  in  young  indi- 
viduals. Previous  heating  or  extraction  with  ether,  according  to  Niki- 
tine,  does  not  prevent  the  action  of  Gram's  stain,  but  treatment  with 
acids  or  alkalies  renders  it  impossible.  Bacteria  so  treated,  however, 
after  one  hour's  immersion  in  Loeffler's  mordant  regain  their  property 
of  staining  with  Gram. 

As  to  the  nature  of  Gram's  staining  solution  it  may  be  mentioned 
that  only  the  pararosanilines  (gentian  violet,  methyl  violet,  arid  Victoria 
blue)  are  suitable  for  the  purpose,  whereas  the  rosanilines  (fuchsin 


M  K  'ROSCOPIC  METHODS  73 

and  methylene  blue)  give  negative  results.  The  reason  for  this  is  that 
the  iodine  compounds  with  the  pararosanilines  are  fast  colors,  while 
those  with  the  rosanilines  are  unstable.  These  latter  compounds  when 
treated  with  alcohol  break  up  into  their  constituents,  the  iodine  is  washed 
out,  and  the  dye  substance  remaining  in  the  tissues  stain  them  uni- 
formly, that  is,  without  differentiation.  But  iodine-pararosaniline  com- 
pounds are  not  thus  broken  up  and  consequently  stain  those  portions 
of  the  tissue  more  or  less,  according  to  the  affinity  which  they  have 
for  the  dye  substance.  The  parts  stained  by  Gram  are  thus  distin- 
guished from  those  stained  violet,  not  only  quantitatively,  but  qualita- 
tively; it  is  not  a  gentian  violet  but  an  iodine-rosaniline  staining  which 
occurs. 

Use  of  Mordants  and  Decolorizing  Agents. — In  films  of  blood  and 
pus,  and  in  tissue  sections,  the  tissue  elements  may  be  stained  to  such 
an  extent  as  to  obscure  the  bacteria.  Hence  many  methods  have  been 
devised  to  use  substances  which,  while  increasing  the  staining  power, 
tend  to  fix  the  stain  in  the  bacteria  and  further  to  treat  by  substances 
which  decolorize  the  overstained  tissue  to  a  greater  or  less  extent  while 
leaving  the  bacteria  stained.  The  staining  capacity  of  a  solution  may 
be  increased  by  (a)  the  addition  of  substances  such  as  carbolic  acid, 
aniline  oil,  or  metallic  salts,  all  of  which  probably  act  as  mordants;  (b) 
by  the  addition  of  weak  alkaline  solutions  of  caustic  potash  or  ammo- 
nium carbonate;  (c)  by  the  employment  of  heat;  (d)  by  long  duration 
of  the  staining  process. 

As  decolorizing  agents  we  use  chiefly  mineral  acids  (hydrochloric, 
nitric,  sulphuric),  vegetable  acids  (acetic),  alcohol  or  a  combination 
of  alcohol  and  acid;  also  various  oils,  aniline,  clove,  etc. 

The  acid  aniline  dyes  are  represented  by  eosin,  acid  fuchsin,  and 
fluorescein. 

Formulae  of  the  Most  Commonly  Used  Stain  Combinations.  LOEFFLER'S 
ALKALINE  METHYLENE-BLUE  SOLUTION.  This  consists  of  concen- 
trated alcoholic  solution  of  methylene  blue,  30  c.c.;  caustic  potash  in 
a  0.01  per  cent,  solution,  100  c.c.  The  alkali  not  only  makes  the  cell 
more  permeable,  but  also  increases  the  staining  power  by  liberating 
the  free  base  from  the  dye. 

KOCH-EHRLICH  ANILINE-WATER  SOLUTION  OF  FUCHSIN  OR  GENTIAN 
VIOLET  is  prepared  as  follows:  To  98  c.c.  of  distilled  water  add  2  c.c. 
aniline  oil,  or  more  roughly  but  with  equally  good  results,  pour  a  few  cubic 
centimetres  of  aniline  oil  into  a  test-tube,  then  add  sufficient  water  to 
nearly  fill  it.  In  either  case  the  mixtures  are  thoroughly  shaken  and 
then  filtered  into  a  beaker  through  moistened  filter-paper  until  the  filtrate 
is  perfectly  clear.  To  75  c.c.  of  the  filtrate  add  25  c.c.  of  the  concen- 
trated alcoholic  solution  of  either  fuchsin,  methylene  blue,  or  gentian 
violet,  or  add  the  alcoholic  solution  until  the  aniline  water  becomes 
opaque  and  a  film  begins  to  form  on  the  surface. 

CARBOLIC  FUCHSIN,  OR  ZIEHL'S  SOLUTION. — Distilled  water,  100  c.c.; 
carbolic  acid  (crystalline),  5  gm.;  alcohol,  10  c.c.;  fuchsin,  1  gm.; 
or  it  may  be  prepared  by  adding  to  a  5  per  cent,  watery  solution  of  car- 


74  PRINCIPLES  OF  BACTERIOLOGY 

bolic  acid  the  saturated  alcoholic  solution  of  fuchsin  until  a  metallic 
lustre  appears  on  the  surface  of  the  fluid.  The  carbolic  acid,  like  the 
alkali,  favors  the  penetration  of  the  stain. 

The  last  two  methods,  combined  with  heating,  are  used  to  stain  spores 
and  certain  resistant  bacteria  intensely,  so  that  they  retain  their  color 
when  exposed  to  decolorizing  agents. 

Carbolic  methylene  blue,  first  used  by  Kiihne,  consists  of  1.5  gm. 
of  methylene  blue,  10  gm.  of  absolute  alcohol,  and  100  c.c.  of  a  5 
per  cent,  solution  of  carbolic  acid.  Carbolic  thionin  consists  of  10 
parts  of  a  saturated  alcoholic  solution  of  thionin  and  100  parts  of  a 
1  per  cent,  solution  of  carbolic  acid. 

GRAM'S  STAIN. — Another  differential  method  of  staining  which  is 
employed  is  that  known  as  Gram's  method.  In  this  method  the  objects 
to  be  stained  are  floated  on  or  covered  with  the  aniline  or  carbolic  gen- 
tian-violet solution.  After  remaining  in  this  for  a  few  minutes  they 
are  rinsed  in  water  and  then  immersed  in  an  iodine  solution  (LugoPs), 
composed  of  iodine,  1  gm.;  potassium  iodide,  2  gm.;  distilled  water, 
300  c.c.  In  this  they  remain  for  from  one  to  three  minutes  and  are 
again  rinsed  in  water.  They  are  then  placed  in  strong  alcohol  until 
most  of  the  dye  has  been  washed  out.  If  the  cover-glass  as  a  whole 
still  shows  a  violet  color,  it  is  again  treated  with  the  iodine  solution, 
followed  by  alcohol,  and  this  is  continued  until  no  trace  of  violet  color 
is  visible  to  the  naked  eye.  They  may  then  be  washed  in  water  and 
examined,  or  a  contrasting  color  of  eosin,  fuchsin,  carmine,  or  Bis- 
marck brown  may  be  given  them  by  inserting  them  in  weak  solutions 
of  these  dyes  for  a  few  minutes.  This  method  is  useful  in  demonstrat- 
ing the  capsule  which  is  seen  to  surround  some  bacteria — particularly 
the  pneumococcus — and  also  in  differentiating  between  varieties  of 
bacteria,  for  some  do  and  others  do  not  retain  their  stain  when  put  in 
the  iodine  solution  for  a  suitable  time. 

Staining  of  Capsules. — Many  methods  of  demonstrating  the  cap- 
sule have  deen  devised.  Two  only  will  be  given  here.  The  glacial 
acetic  acid  method,  as  described  by  Welch,  is  as  follows:  1.  Cover  the 
preparation  with  glacial  acetic  acid  for  a  few  seconds.  2.  Drain 
off  and  replace  with  aniline  gentian-violet  solution;  this  is  to  be  repeat- 
edly added  until  all  the  acid  is  replaced.  3.  Wash  in  1  to  2  per  cent, 
solution  of  sodium  chloride  and  mount  in  the  same. 

Hiss'  COPPER  SULPHATE  METHOD. — The  organisms  are  grown,  if 
possible,  on  ascitic  fluid  or  serum  media.  If  not,  spread  the  organisms 
on  the  cover-glass  by  mixing  with  a  drop  of  serihn,  or,  better,  a  drop 
of  one  of  the  diluted  serum  media.  Dry  in  the  air  and  fix  by  heat. 

The  capsules  are  stained  by  the  following  method:  A  5  per  cent,  or 
10  per  cent,  aqueous  solution  of  gentian  violet  or  fuchsin  (5  c.c.  saturated 
alcoholic  solution  gentian  violet  to  95  c.c.  distilled  water)  is  used.  This 
is  placed  on  the  dried  and  fixed  cover-glass  preparation  and  gently 
heated  for  a  few  seconds  until  steam  arises.  The  dye  is  washed  off 
with  a  20  per  cent,  solution  of  copper  sulphate  (crystals).  The  prep- 
aration is  then  placed  between  filter-paper  and  thoroughly  dried. 


MICROSCOPIC  METHODS  75 

Staining  Spores. — We  have  already  noted  that  during  certain 
stages  in  the  growth  of  a  number  of  bacteria  spores  are  formed  which 
refuse  to  take  up  color  when  the  bacteria  are  stained  in  the  ordinary 
manner.  Special  methods  have  been  devised  for  causing  the  color  to 
penetrate  through  the  resistant  spore.  In  the  simplest  method  a  cover- 
slip  after  having  been  prepared  in  the  usual  way  is  covered  with  Ziehl's 
carbolic  fuchsin  solution  and  held  over  the  Bunsen  flame  until  the 
fluid  steams.  This  is  continued  for  one  or  two  minutes.  It  is  then 
washed  and  dipped  in  a  decolorizing  acid  solution,  such  as  a  2  per 
cent,  alcoholic  solution  of  nitric  acid,  or  a  1  per  cent,  solution  of 
sulphuric  acid  in  water,  until  all  visible  color  has  disappeared,  then 
it  is  washed  off  and  dipped  for  one-half  minute  in  a  saturated  watery 
solution  of  methylene  blue.  The  bacilli  will  then  be  blue  and  the 
spores  red.  Sometimes,  however,  the  spores  refuse  to  take  the  stain  in 

FIG.  52 


*• 

V 


«.       •*    *l    - 

•     ••v:H    $ 

-,•'->  ••-     •'•*!" 


Culture  stain  by  Hiss'  method. 

this  manner.  We  then  can  adopt  Moeller's  method,  which  is  designed 
still  further  to  favor  the  penetration  of  the  coloring  matter  through  the 
spore  membrane.  The  prepared  cover-slip  is  held  for  two  minutes  in 
chloroform,  then  washed  off  in  water,  then  placed  from  one-half  to 
three  minutes  in  a  5  per  cent,  solution  of  chromic  acid,  again  washed 
off  in  water,  and  now  stained  by  adding  to  it  carbolic  fuchsin,  which 
is  steamed  for  several  minutes.  The  staining  fluid  is  then  washed  off 
and  the  preparation  decolorized  in  a  3  per  cent,  solution  of  hydrochloric 
acid  or  a  5  per  cent,  solution  of  sulphuric  acid.  The  preparation  is 
finally  stained  for  a  minute  in  methylene-blue  solution.  The  spores 
will  be  red  and  the  body  of  the  cells  blue.  The  different  spores  vary 
greatly  in  the  readiness  with  which  they  take  up  the  dyes,  and  we  have, 
therefore,  to  experiment  with  each  variety  as  to  the  length  of  time  they 


76  PRINCIPLES  OF  BACTERIOLOGY 

should  be  exposed  to  the  maceration  of  the  chromic  acid.  Even  under 
the  best  conditions  it  is  almost  impossible  to  stain  some  spores. 

Staining  Flagella. — For  the  demonstration  of  flagella,  which  are 
possessed  by  all  motile  bacteria,  we  are  indebted  to  Loeffler.  The 
staining  of  flagella  satisfactorily  is  one  of  the  most  difficult  of  bacterio- 
logical procedures.  Special  stains  devised  by  him,  by  Van  Ermengem, 
by  Pitfield,  and  others  are  employed. 

Preparation  of  Films. — In  all  methods  young  (twelve  to  eighteen-hour) 
cultures  on  agar  should  be  chosen.  A  little  of  the  culture  is  carefully 
removed  and  placed  in  a  few  drops  of  filtered  tap  water.  A  tiny  drop 
of  this  rather  thin  emulsion  is  allowed  to  spread  with  as  little  manipu- 
lation as  possible  over  the  cover-glass. 

Bunge's  modification  of  Loeffler's  method  is  carried  out  as  follows: 
Cover-glasses  which  have  been  most  carefully  cleaned  are  covered  by  a 
very  thin  smear.  After  drying  in  the  air  and  passing  three  times  through 
the  flame  the  smear  is  treated  with  a  mordant  solution,  which  is  pre- 
pared as  follows:  To  3  parts  of  saturated  watery  solution  of  tannin 
add  1  part  of  a  25  per  cent,  solution  of  ferric  chloride.  This  mordant 
should  be  allowed  to  stand  for  several  weeks  before  using.  After  pre- 
paring the  cover-slip  with  all  precautions  necessary  to  cleanliness  the 
filtered  mordant  is  allowed  to  act  cold  for  five  minutes,  after  which  it 
is  warmed  and  then  in  one  minute  washed  off.  After  drying,  the 
smear  is  stained  with  the  carbol-fuchsin  solution  or  carbol-gentian 
violet,  and  then  washed  off,  dried,  and  mounted. 

Frequently  the  flagella  appear  well  stained,  but  often  the  process  has 
to  be  repeated  a  number  of  times.  Overheating  of  the  film  prevents  the 
staining  of  the  flagella. 

Van  Ermengem 's  method  gives  good  results. 

Examination  of  Bacteria  in  Tissues. — Occasionally  it  is  of  importance  to 
examine  the  bacteria  as  they  are  in  the  tissues  themselves.  The  tissues 
should  be  obtained  soon  after  death,  so  as  to  prevent  as  much  as  possible 
post-mortem  changes,  with  consequent  increase  or  decrease  in  the 
number  of  bacteria  in  them.  Selected  pieces  of  tissues  can  be  frozen  by 
ether  and  sections  cut,  but  the  best  results  are  obtained  when  the  material 
is  embedded  in  celloidin  or,  better  still,  in  paraffin.  From  the  properly 
selected  spots  small  portions,  not  thicker  than  one-quarter  of  an  inch, 
are  removed  and  placed  in  absolute  alcohol  for  one  or  two  days  if  less 
than  one-eighth  inch  thick,  and  longer  if  thicker.  For  the  larger  pieces 
it  is  better  to  change  the  aclohol  after  twenty-four  hours.  The  tissue 
pieces  should  be  kept  from  falling  to  the  bottom  as  the  higher  layers 
of  alcohol  remain  nearer  absolute.  If  along  with  the  bacteria  one  wishes 
to  study  the  finer  structure  of  the  tissue  we  must  employ  formalin  or 
corrosive  sublimate.  The  tissue  is  put  in  formalin  4  to  10  percent,  solution 
for  three  to  twenty-four  hours,  and  then  in  alcohol.  Corrosive  sublimate 
as  a  saturated  solution  in  0.75  per  cent,  sodium  chloride  solution  is  also 
an  excellent  fixative  agent.  Dissolve  the  sublimate  in  the  salt  solution 
by  heat  and  then  allow  to  cool,  when  the  separation  of  crystals  will 
show  that  saturation  is  complete.  For  pieces  of  tissue  one-eighth  inch 


MICROSCOPIC  METHODS  77 

in  thickness  twelve  hours'  immersion  is  sufficient,  if  larger  twenty-four 
hours  is  necessary.  They  should  then  be  placed  in  pieces  of  gauze 
and  left  in  running  water  for  from  twelve  to  twenty-four  hours,  accord- 
ing to  the  size  of  the  pieces,  to  wash  out  the  excess  of  sublimate.  They 
are  then  placed  for  twenty-four  hours  in  each  of  the  following  strengths 
of  methylated  spirit  (free  from  naphtha):  30  per  cent.,  60  per  cent., 
and  90  per  cent.  Finally  they  are  placed  in  absolute  alcohol  for  twenty- 
four  hours  and  are  then  ready  to  be  prepared  for  cutting  according 
to  the  usual  histological  methods.  The  paraffin  sections  of  tissue  having 
been  prepared  and  cut,  they  are  ready  for  staining.  t 

LOKFFLKR'S  METHOD. — The  section  is  placed  in  Loeffler's  alkaline 
methylene-blue  solution  for  5  to  30  minutes,  then  placed  for  a  few  sec- 
onds in  1  per  cent,  acetic  acid.  It  is  then  placed  in  absolute  alcohol, 
xylol,  and  Canada  balsam.  The  number  of  seconds  during  which  the 
preparation  remains  in  the  acetic  acid  must  be  tested  by  trials.  The 
bacteria  are  dark  blue,  the  nuclei  blue,  and  the  cell  bodies  light 
blue. 

Thionin  solution,  carbol-fuchsin  solution,  and  gentian  violet  can  be 
used  instead  of  Loeffler's  methylene  blue.  Gram's  method,  with  3 
per  cent,  hydrochloric  acid  in  alcohol  as  a  tissue  decolorizer  for  ten 
seconds,  is  also  valuable. 

Preservation  of  Specimens. — Dry  unstained  or  stained  preparations 
of  bacteria  keep  indefinitely,  but  if  mounted  in  Canada  balsam,  cedar 
oil,  or  dammar  lac  they  tend  to  gradually  fade,  but  may  be  preserved 
for  many  months  or  vears. 


THE  MICROSCOPE   AND   THE  MICROSCOPIC   EXAMINATION 
OF    BACTERIA. 

Different  Parts  of  the  Microscope. — A  complete  instrument  usually 
has  four  oculars,  or  eye-pieces  (^4),  which  are  numbered  from  1  to  4, 
according  to  the  amount  of  magnification  which  they  yield.  Numbers 
2  and  4  are  most  useful  for  bacteriological  work.  The  objective — the 
lens  at  the  distal  end  of  the  barrel — serves  to  give  the  main  magnifica- 
tion of  the  object.  For  stained  bacteria  the  y1^  achromatic  oil-immer- 
sion lens  is  regularly  employed;  except  for  photographic  purposes  the 
apochromatic  lenses  are  not  needed.  Even  here  they  are  not  indis- 
pensable. A  T%  may  at  times  be  useful,  but  hardly  necessary;  a  No. 
4  ocular  and  a  -^  lens  give  a  magnification  of  about  1000  diameters 
(Fig.  55).  For  unstained  bacteria  we  employ  either  the  TV  immersion 
or  y  dry  lens,  according  to  the  purpose  for  which  we  study  the  bac- 
teria; for  the  examination  of  colonies  where,  as  a  rule,  we  do  not  wish 
to  see  individual  bacteria,  but  only  the  general  appearance  of  whole 
groups,  we  use  lenses  of  much  lower  magnification  (Fig.  56). 

The  stage  C — the  platform  upon  which  the  object  rests — should  be 
large  enough  to  support  the  Petri  plates  if  culture  work  is  to  be  done. 
The  iris  diaphragm  D,  which  is  now  regularly  used  in  bacteriological 
work,  opens  and  closes  like  the  iris  of  the  eye,  and  so  controls  the 


78 


PRINCIPLES  OF  BACTERIOLOGY 


amount  of  light.  Its  opening  is  diminished  or  increased  by  moving  a 
small  arm,  which  is  underneath  the  stage,  in  one  or  another  direction. 
The  reflector  placed  beneath  the  stage  serves  to  direct  the  light  to  the 
object  to  be  examined.  It  has  two  surfaces — one  concave  and  one 
convex.  The  coarse  adjustment  F  is  the  rack-and-pinion  arrange- 
ment by  which  the  barrel  of  the  microscope  can  be  quickly  raised  or 
lowered.  It  is  used  to  bring  the  bacteria  roughly  into  focus.  If  the 


FIG.  53 


FIG.  54 


-—Eye-lens 


'Body  Tube 


of  the  Ocular 


Draw  Tube  Diaphragm 
with  Society  Screw 


--Society  Screw 


.--Mount 

Back-lens 
Middle-lens 
Front-lens 
Working  Distance 


ofthe 
Objective 


Microscope. 


Slide-" 
Object' 


Internal  structure  of  the  microscope. 
(After  Spencer  Lens  Co.) 


bearings  become  loose  tighten  the  little  screws  at  the  back  of  the  pinion 
box.  Keep  the  teeth  clean.  If  the  bearings  need  oiling  use  an  acid-free 
lubricant,  such  as  paraffin  oil.  The  fine  adjustment  G  serves  to  raise 
and  lower  the  barrel  very  slowly  and  evenly,  and  is  used  for  the  exact 
study  of  the  bacteria  when  high-power  lenses  are  used.  It  is  neces- 
sarily of  limited  range  and  delicate  in  its  mechanism.  If,  when  look- 


MICROSCOPIC  METHODS 


79 


ing  into  the  eye-piece,  no  change  of  focus  is  noticed  1>\  turning  the 
micrometer  head,  or  if  the  micrometer  head  ceases  to  turn,  the  adjust- 
ment has  reached  its  limit.  Turn  the  micrometer  back  to  bring  the 
fine  adjustment  midway  within  its  range.  When  the  fine  adjustment 
head  stops  do  not  force  it.  For  the  microscopic  study  of  bacteria 
it  is  essential  that  we  magnify  the  bacteria  as  much  as  possible  and 
still  have  their  definition  clear  and  sharp.  It  is  essential,  therefore, 
that  the  microscope  be  provided  with  an  oil-immersion  system  and  a 
substage  condensing  apparatus.  In  using  the  oil-immersion  lens  a 
drop  of  oil  of  the  same  index  of  refraction  as  the  glass  is  placed  upon 
the  face  of  the  lens,  so  as  to  connect  it  with  the  cover-glass  when  the 
bacteria  are  in  focus.  There  is  thus  no  loss  of  sight  through  deflection, 
as  is  the  case  in  the  dry  system.  If  the  lenses  become  dirty  they  should 
be  wiped  gently  with  Japanese  lens  paper  or  a  clean,  soft,  old-linen 
handkerchief.  If  necessary  breathe  on  the  lens  before  wiping,  and  if 


FIG.  55 


FIG.  56 


Anthrax  bacilli  and  blood  cells. 
X  1000  diameters. 


Colonies  of  diphtheria  bacilli. 
X  200  diameters. 


this  does  not  succeed  use  a  little  xylol  or  chloroform.  These  substances 
are  not  to  be  used  unless  necessary.  An  immersion  objective  should 
always  be  cleaned  immediately  after  using.  The  objective  should 
always  be  kept  covered  so  as  to  prevent  dust  dropping  in. 

Light. — The  best  light  is  obtained  from  white  clouds  or  a  blue  sky. 
Avoid  direct  sunlight.  If  necessary  use  white  shades  to  modify  the 
sunlight.  Artificial  light  has  one  advantage  over  daylight  in  that  it  is 
constant  in  quality  and  quantity.  The  Welsbach  burner  and  a  whitened 
incandescent  bulb  give  a  good  light.  A  blue  glass  between  the  artificial 
light  and  the  lens  is  often  of  value.  An  eye-shade  is  often  helpful. 

Substage  Condensing  Apparatus  //  is  a  system  of  lenses  situated 
beneath  the  central  opening  of  the  stage.  It  serves  to  condense  the 
light  passing  through  the  reflector  to  the  object  in  such  a  wray  that  it  is 
focused  upon  the  object,  thus  furnishing  the  greatest  amount  of  lumi- 
nosity. Between  the  condenser  and  the  reflector  is  placed  an  adjustable 


80  PRINCIPLES  OF  BACTERIOLOGY 

diaphragm,  the  aperture  of  which  can  be  regulated,  as  circumstances 
require,  to  permit  of  either  a  very  small  or  a  very  large  amount  of  light 
passing  to  the  object. 

Focusing. — Focus  the  body  tube  down  by  means  of  the  coarse  adjust- 
ment until  the  objective  nearly  touches  the  cover-glass,  being  careful 
not  to  touch  it.  Then  with  the  eye  at  the  eye-piece  focus  up  carefully 
with  the  coarse  adjustment  until  the  specimen  comes  plainly  into  view. 
Be  careful  not  to  pass  by  this  focal  point  without  noticing  it.  This  is 
likely  to  occur  if  the  light  be  too  intense  and  the  specimen  thin  and 
transparent.  If  the  sliding  tube  coarse  adjustment  is  used,  focus  care- 
fully by  giving  the  tube  a  spiral  movement. 

When  the  object  is  brought  fairly  well  into  focus  by  means  of  the 
coarse  adjustment,  use  the  fine  adjustment  to  focus  on  the  particular 
spot  desired,  for  if  this  spot  is  in  the  centre  of  the  field  of  the 
low  power  it  will  be  somewhere  in  the  field  of  the  higher  power.  It  is 
too  much  to  ask  of  the  maker  that  the  lenses  be  made  absolutely  par- 
focal  and  centred.  The  delicacy  of  the  centring  can  be  appreciated 
when  the  magnification  and  the  extremely  small  portion  examined  are 
considered.  When  the  objectives  are  not  thus  fitted  to  the  nose-piece, 
refocusing  and  again  hunting  up  the  object  are  necessary.  In  so  doing 
we  repeat  the  caution  to  always  focus  up  before  turning  the  nose- 
piece.  When  no  revolving  nose-piece  is  used  the  change  of  objectives 
means  the  unscrewing  of  one  and  the  screwing  of  the  other  into  its 
place,  and  refocusing. 

The  beginner  should  always  use  the  low-power  objectives  and  oculars 
first.  The  low-power  objectives  have  longer  working  distances  and 
are  not  so  apt  to  be  injured.  They  always  show  a  larger  portion  of  the 
specimen  and  thus  give  one  a  better  idea  of  the  general  contour.  After 
obtaining  this  general  idea  the  higher  powers  can  be  used  to  bring  out 
greater  detail  in  any  particular  part. 

Tube  Length  and  Cover-glass. — All  objectives  are  corrected  to  a  cer- 
tain tube  length  (160  mm.  by  most  makers — Leitz,  170  mm.)  and  all 
objectives  in  fixed  mounts  of  over  0.70  N.  A.  are  corrected  to  a  definite 
thickness  of  cover-glass  as  well  (Zeiss,  0.15  mm.,  0.20  mm.;  Leitz,  0.17 
mm.;  Bausch  &  Lomb  and  Spencer,  0.18  mm.).  These  objectives 
give  their  best  results  only  when  used  with  the  cover-glass  and  tube 
length  for  which  they  are  corrected.  As  indicated  in  Fig.  53  the 
tube  length  extends  from  the  eye  lens  of  the  eye-piece  to  the  end  of 
the  tube  into  which  the  objective  or  nose-piece  is  screwed.  If  a  nose- 
piece  is  used  the  draw  tube  must  be  correspondingly  shortened.  If 
the  cover-glass  is  thinner  than  that  for  which  the  objective  is  cor- 
rected, the  tube  must  be  lengthened  to  obtain  best  results;  if  thicker, 
shortened. 

The  more  expensive  objectives  are  provided  with  adjustable  mounts 
by  which  the  distances  between  the  lens  systems  may  be  changed  to 
compensate  for  difference  of  thickness  of  cover.  They  are  success- 
fully used  only  in  the  hands  of  an  expert.  One  of  them' out  of  adjust- 
ment is  worse  than  an  ordinary  objective. 


MICROSCOPIC  METHODS  81 

Examination  of  Bacteria  injthe  Hanging  Drop. — It  is  often  valuable  to 
observe  bacteria  alive,  so  as  to  study  them  under  natural  conditions. 
\Y<-  can  thus  note  the  method  and  rate  of  their  multiplication,  the 
pivsi'iice  or  absence  of  spore  formation,  and  their  motility  in  fluids  and 
their  agglutination  with  specific  serums.  For  this,  special  slides  and 
methods  are  desirable.  The  usual  form  is  one  in  which  there  is  ground 
out  on  one  surface  a  hollow  having  a  diameter  of  about  half  an  inch 
(Fig.  57).  According  to  the  purpose  for  which  the  hanging  drop  is  to 


FIG.  57 


Hollow  slide  with  cover-glass. 

be  studied,  sterilization  of  the  slide  and  cover-glass  may  or  may  not 
be  necessary.  The  hanging  block  has  already  been  described.  The 
technique  of  preparing  and  studying  the  hanging  drop  is  as  follows :  The 
surface  of  the  glass  around  the  hollow  in  the  slide  is  smeared  with  a 
little  vaselin  or  other  inert  substance.  This  has  for  its  purpose  both 
the  sticking  of  the  cover-glass  to  the  slide  and  the  prevention  of 
evaporation  in  the  drop  placed  in  the  little  chamber,  which  is  to  be 
formed  between  the  cover-glass  when  placed  over  the  hollow,  and  the 
slide. 

For  the  purpose  of  studying  the  bacteria  we  place,  if  they  are  in 
fluids,  simply  a  platinum  loopful  upon  the  centre  of  the  cover-glass  and 
then  invert  it  by  means  of  a  slender  pair  of  forceps  over  the  hollow  in  the 
slide,  being  very  careful  to  have  the  bacteria  over  the  very  centre  of  the 
space.  If  the  bacteria,  on  the  contrary,  are  growing  on  solid  media,  or  are 
obtained  from  thick  pus  or  tissues  from  organs,  they  are  mixed  with  a 
suitable  amount  of  bouillon  or  sterile  physiological  salt  solution  either 
before  or  after  being  placed  upon  the  cover-glass.  If  we  wish  to  observe 
the  bacteria  under  natural  conditions  we  must  keep  the  tiny  drop  of  fluid 
at  the  proper  temperature  for  the  best  growth  of  the  bacteria.  If, 
however,  we  simply  wish  to  observe  their  form  and  arrangement  this  is 
not  necessary. 

In  the  study  of  living  bacteria  we  often  wish  to  observe  their  group- 
ing and  motion  rather  than  their  individual  characters,  and  so  use  less 
magnification  than  for  stained  bacteria.  In  studying  unstained  bac- 
teria and  tissues  we  shut  off  as  large  a  portion  of  the  light  with  our 
diaphragm  as  is  compatible  with  distinct  vision,  and  thus  favor  con- 
trasts which  appear  as  lights  and  shadows,  due  to  the  differences  in 
light  transmission  of  the  different  materials  under  examination.  It  is 
necessary  to  remember  thai  they  are  seen  with  difficulty,  and  that  we 
are  very  apt,  unless  extremely  careful  in  focusing,  to  allow  the  lens 
to  go  too  far,  and  so  come  upon  the  cover-glass,  break  it,  destroy  our 
preparation,  and,  if  examining  parasitic  bacteria,  infect  the  lens.  This 
may  be  avoided  by  first  finding  the  hanging  drop  with  a  low-power  lens 
and  thus  exactly  centre  it.  The  lens  of  higher  magnification  is  now 

6 


82 


PRINCIPLES  OF  BACTERIOLOGY 


very  gradually  lowered,  while  at  the  same  time  gently  moving  the  slide 
back  and  forth  to  the  slightest  extent  possible  with  the  left  hand.  If 
any  resistance  is  felt  the  lens  should  be  raised,  for  it  has  gone  beyond 
the  point  of  focus  and  is  touching  the  cover-glass. 

Testing  Agglutinative  Properties  of  Serum. — By  agglutination  is  meant 
the  aggregation  into  clumps  of  uniformly  disposed  bacteria  in  a  fluid; 
by  sedimentation  the  formation  of  a  deposit  composed  of  such  clumps 
when  the  fluid  is  allowed  to  stand;  sedimentation  is  thus  the  naked-eye 
evidence  of  agglutination. 

The  blood  serum  of  animals  is  found  to  acquire  the  clumping  power 
for  almost  every  variety  of  motile  bacteria,  and  for  many  non-motile 
forms  after  infection  with  that  variety. 

In  order  to  help  the  student  to  thoroughly  understand  what  com- 
prises a  reaction,  Wilson  prepared  a  set  of  drawings,  which  are  here 
reproduced.  The  culture  to  be  tested  should  be  of  about  twenty  hours' 


FIG.  58 


FIG.  59 


Microscopic  field,  showing  the  top  of  a  hanging  •  ;  Microscopic  field,  showing  a  cross-section  of  the 
drop  in  a  normal  typhoid  culture.  drop  in  Fig.  58. 


growth,  either  in  bouillon  or  on  agar.  If  on  the  latter  a  suspension 
is  made  in  broth  or  normal  salt  solution.  A  loopful  of  the  fluid  con- 
taining the  bacteria  is  placed  on  the  cover-glass,  and  to  it  an  equal 
quantity  of  the  serum  dilution  is  added. 

In  making  the  hanging  drop  to  be  examined  it  is  necessary  to  have 
it  of  such  a  depth  that  it  will  show  at  least  three  focal  planes,  otherwise 
the  examination  will  be  incomplete  and  unsatisfactory.  The  moist 
chamber  must  be  well  sealed  by  vaselin  so  as  to  prevent  drying,  and 
kept  at  a  temperature  of  at  least  20°  and  not  over  35°  C. 

Fig.  58  shows  a  microscopic  field  of  the  top  of  a  hanging  drop 
of  a  normal  bouillon  culture  of  typhoid  bacilli.  The  culture  is  twenty 
hours  old  and  the  organisms  are  freely  motile.  This  represents  the 
control  drop  used  for  comparison  with  the  drop  of  the  same  culture, 


MICROSCOPIC  METHODS 


83 


to  which  has  been  added  a  little  of  the  blood  of  a  person  suspected  to 
have  typhoid.  Note  these  points  in  Fig.  58:  the  organisms  are  evenly 
distributed  throughout  the  field,  except  at  the  edge  of  the  drop,  where 
they  are  gathered  in  great  numbers;  they  show  great  activity  here, 
seemingly  trying  to  crowd  to  the  very  edge.  This  attraction  is  probably 
due  to  the  action  exerted  on  the  organisms  by  the  oxygen  in  the  air, 
which  naturally  exerts  positive  chemotaxis  on  all  aerobic  organisms. 

Fig.  59  shows  a  cross-section  of  the  drop  represented  in  Fig.  58,  and 
it  will  be  noticed  that  the  bacilli  are  evenly  distributed  throughout  the 
drop,  except  at  one  place  in  the  focal  plane  a,  and  again  in  the  focal 
plane  c. 

It  sometimes  happens  that  there  is  a  substance  adhering  to  a  sup- 
posedly clean  cover-glass  which  attracts  the  bacilli  to  that  point,  where 


FIG.  60 


FIG.  61 


Microscopic  field,  showing  the  top  of  a  drop  with 
the  typhoid  reaction. 


Microscopic  field,  showing  a  cross-section  of 
the  drop  in  Fig.  60. 


they  appear  as  fairly  well-defined  clumps,  more  or  less  like  the  true 
clumps  due  to  the  agglutinating  substance  in  typhoid  blood.  The 
increase  in  organisms  at  the  bottom  of  the  drop  in  the  focal  plane  c  is 
easily  accounted  for  by  the  fact  that  gravity  naturally  carries  the  dead 
and  non-motile  organisms  to  the  bottom,  these  frequently  assuming  the 
character  of  clumps. 

If  a  field  can  be  found  in  any  focal  plane  of  the  hanging  drop  free 
from  clumps,  one  can  be  quite  sure  that  any  clumping  present  is  not 
due  to  any  agglutinating  substance  which  necessarily  will  affect 
organisms  in  every  focal  plane. 

Fig.  60  shows  the  microscopic  appearance  of  the  top  of  a  drop 
where  the  reaction  is  present.  Notice  first  that  the  organisms  have  been 
drawn  together  in  groups  and  that  the  individuals  of  each  group  appear 
to  be  loosely  held  together.  Viewed  under  the  microscope  these  clumps 
are  practically  quiescent,  there  being  very  little  movement  either  of  the 
individual  organisms  or  of  the  clump  as  a  whole.  The  edge  of  the  drop 


84 


PRINCIPLES  OF  BACTERIOLOGY 


is  practically  free  from  organisms,  showing  that  the  air  no  longer  exerts 
any  influence  on  them. 

Fig.  61  shows  a  cross-section  of  the  hanging  drop  shown  in  Fig.  60. 
The  clumps  are  evenly  distributed  throughout  the  drop,  with  perhaps 
some  increase  in  the  numbers  and  compactness  of  the  clumps  at  the 
bottom. 

Fig.  62  shows  the  microscopic  appearance  of  the  top  of  a  hanging 
drop  of  a  bouillon  culture  to  which  has  been  added  some  blood  of  a 
patient  suffering  from  a  febrile  condition  not  caused  by  typhoid  infec- 
tion, but  which  exerts  a  slight  non-specific  influence  on  the  typhoid 
organisms.  It  will  be  seen  that  the  reaction  is  incomplete  and  that 
there  are  many  organisms  at  the  edge  of  the  drop.  The  air  exerts  the 
same  influence  on  the  bacilli  that  it  did  before  the  addition  of  the 
blood.  Note  the  character  of  the  clumps,  generally  small  and  com- 
pact at  the  centre,  with  the  bacilli  at  the  edge  of  the  clump,  usually 
attached  by  one  end  only. 


FIG.  62 


FIG.  63 


Microscopic  field,  showing  the  top  of  a  drop  of 
culture  with  reaction  not  due  to  typhoid. 


Microscopic  field,  showing  a  cross-section 
of  Fig.  62. 


Very  frequently  these  clumps  have  the  appearance  of  being  built] 
up  around  a  piece  of  detritus  present  in  the  clump.  All  the  organisms 
comprising  the  clump  seem  to  have  retained  part,  at  least,  of  their 
motility,  those  on  the  edges  being  particularly  motile,  so  far  as  their 
free  ends  are  concerned. 

When  motility  is  very  much  inhibited  these  clumps  have  a  peculiar 
trembling  movement,  which  is  like  the  molecular  movement  described 
by  Brown. 

Fig.  63  shows  a  cross-section  of   the  drop  represented  in  Fig.  62.1 
Note  the  same  character  of  the  clumps  in  every  focal  plane:  the  large 
number  of  motile  bacilli  and  the  number  attracted  to  the  edge  of  the 
drop  by  the  air. 


MICROSCOPIC  METHODS 


85 


Important    Characteristics    trhich    xhould   be  Noted    in   the 
Study    of   a    Bacterium. — The    accompanying    chart    gives    the    most 
important  points  to  be  investigated.     With  some  varieties  the  cultural 


At;  robe 


Growth  at  37 


:»I;..\.  Growth 


Glucose 


Growth  in 

clo-rd  arm 


Odor 


Chromogenic 


liacillu? 


O>;vus 


Threads  or 
Chains 


I    I 


Gram. 


Nitrate  Reduc. 


Indol 


Pathogeuicity 


Sediment 


Lique- 
faction 


Surtao- 


Agglutination 
characteristics 


Bactericidal 
proi" 


Soluble 

toxins 


Color 


Coairulat' 

id 


Type  of  card  used  in  studying  characteristics  of  bacteria. 

characteristics  are  of  the  greatest  importance,  while  with  others  it  is 
the  study  of  pathogenic  or  toxic  effects. 


CHAPTER  VII. 

VITAL  PHENOMENA  OF  BACTERIA— MOTION,  HEAT,  AND  LIGHT 
PRODUCTION— CHEMICAL  EFFECTS. 

Motility. — Many  bacteria  when  examined  under  the  microscope  are 
seen  to  exhibit  active  movements  in  fluids.  The  movements  are  of  a 
varying  character,  being  described  as  rotary,  undulatory,  sinuous,  etc. 
At  one  time  they  may  be  slow  and  sluggish,  at  another  so  rapid  that 
any  detailed  observation  is  impossible.  Some  bacteria  are  very  active 
in  their  movements,  different  individuals  progressing  rapidly  in  differ- 
ent directions,  while  with  many  it  is  difficult  to  say  positively  whether 
there  is  any  actual  motility  or  whether  the  organism  shows  only  molec- 
ular movements — so-called  "Brownian"  movements — a  dancing,  trem- 
bling motion  possessed  by  all  finely  divided  organic  particles.  Very 
young  cultures  in  neutral  nutrient  bouillon  should  be  examined  at  a 
temperature  suitable  for  their  best  growth.  Not  all  species  of  bacteria 
which  have  flagella  exhibit  at  all  times  spontaneous  movements;  such 
movements  may  be  absent  in  certain  culture  media  and  at  too  low  or 
too  high  temperatures,  or  of  either  an  insufficient  or  excessive  supply 
of  oxygen. 

Chemotaxis. — Some  chemical  substances  seem  to  exert  a  peculiar 
attraction  for  bacteria,  known  as  positive  chemotaxis,  while  others  repel 
them — negative  chemotaxis.  Moreover,  all  varieties  are  not  affected 
alike,  for  the  same  substances  may  exert  on  some  bacteria  an  attraction 
and  on  others  a  repulsion.  Oxygen,  for  example,  attracts  aerobic  and 
repels  anaerobic  bacteria,  and  for  each  variety  there  is  a  definite  propor- 
tion of  oxygen,  which  most  strongly  attracts.  The  chemotactic  proper- 
ties of  substances  are  tested  by  pushing  the  open  end  of  a  fine  capillary 
tube,  filled  with  the  substance  to  be  tested,  into  the  edge  of  a  drop  of 
culture  fluid  containing  bacteria  and  examining  the  hanging  drop 
under  the  microscope.  We  are  able  thus  to  watch  the  action  of  the 
bacteria  and  note  whether  they  crowd  about  the  tube  opening  or  are 
repelled  from  it.  Among  substances  showing  positive  chemotaxis  for 
nearly  all  bacteria  are  peptone  and  urea,  while  among  those  showing 
negative  chemotaxis  are  alcohol  and  many  of  the  metallic  salts. 

Production  of  Light. — Bacteria  which  have  the  property  of  emitting 
light  are  quite  widely  distributed  in  nature,  particularly  in  media  rich 
in  salt,  as  in  sea-water,  salt  fish,  etc.  Many  of  these,  chiefly  bacilli  and 
spirilla,  have  been  accurately  studied.  The  emission  of  light  is  a  prop- 
erty of  the  living  protoplasm  of  the  bacteria,  and  is  not  usually  due  to 
the  oxidation  of  any  photogenic  substance  given  off  by  them;  at  least 
only  in  two  instances  has  such  substance  been  claimed  to  have  been 


VITAL  PIIKXOMEXA  OF  BACTERIA  87 

isolated.  Every  agent  which  is  injurious  to  the  existence  of  the  bacteria 
affects  this  property.  Living  bacteria  are  always  found  in  phosphor- 
escent cultures;  a  filtered  culture  free  from  germs  is  invariably 
non-phosphorescent;  but  while  the  organism  cannot  emit  light  except 
during  life,  it  can  live  without  emitting  light,  as  in  an  atmosphere  of 
carbonic  acid  gas,  for  instance.  They  are  best  grown  under  free  access 
of  oxygen  in  a  culture  medium  prepared  by  boiling  fish  in  sea-water 
(or  water  containing  3  per  cent,  sea-salt),  to  which  1  per  cent,  peptone, 
1  per  cent,  glycerin,  and  0.5  per  cent,  asparagin  are  added.  Even  in 
this  medium  the  power  of  emitting  light  is  soon  lost  unless  the  organ- 
ism is  constantly  transplanted  to  fresh  media. 

Thermic  Effects. — The  production  of  heat  by  bacteria  does  not  attract 
attention  in  our  usual  cultures  because  of  its  slight  amount,  and  even 
fermenting  culture  liquids  with  abundance  of  bacteria  cause  no  sensa- 
tion of  warmth  when  touched  by  the  hand.  Careful  tests,  however, 
show  that  heat  is  produced.  The  increase  of  temperature  in  organic 
substances  when  stored  in  a  moist  condition,  as  tobacco,  hay,  manure, 
etc.,  is  one  partly  at  least  due  to  the  action  of  bacteria. 

Chemical  Effects. — The  processes  which  bodies  being  split  up  undergo 
depend,  first,  on  the  chemical  nature  of  the  bodies  involved  and  the 
conditions  under  which  they  exist,  and,  secondly,  on  the  varieties  of 
bacteria  present.  A  complete  description  of  these  chemical  changes 
is  at  present  impossible.  Chemists  can  as  yet  only  enumerate  some  of 
the  final  substances  evolved,  and  describe,  in  a  few  cases,  the  manner 
in  which  they  were  produced.  Bacteria  are  able  to  construct  their  body 
substance  out  of  various  kinds  of  nutrient  materials  and  also  to  pro- 
duce fermentation  products  or  poisons,  and  they  are  able  to  do  these 
things  either  analytically  or  synthetically  with  almost  equal  ease.  This 
ambidextrous  metabolic  power  exists,  according  to  Hueppe,  among 
bacteria  to  an  extent  known  as  yet  among  no  other  living  things. 

In  the  chemical  building  up  of  their  body  substance  we  can  distin- 
guish, as  Hueppe  concisely  puts  it,  several  groups  of  phenomena: 
Polymerization,  a  sort  of  doubling  up  of  a  simple  compound;  synthesis, 
a  union  of  different  kinds  of  simple  compounds  into  one  or  more  com- 
plex substances;  formation  of  anhydride,  by  which  new  substances 
arise  from  a  compound  through  the  'loss  of  water;  and  reduction  or 
loss  of  oxygen,  which  is  brought  about  especially  by  the  entrance  of 
hydrogen  into  the  molecule.  The  breaking  down  of  organic  bodies  of 
complicated  molecular  structure  into  simpler  combinations  takes  place, 
on  the  other  hand,  through  the  loosening  of  the  bonds  of  polymeriza- 
tion, through  hydration  or  entrance  of  water  into  the  molecule,  and 
through  oxidation. 

The  chemical  effects  which  take  place  from  the  action  of  bacteria 
are  greatly  influenced  by  the  presence  or  absence  of  free  oxygen.  The 
access  of  pure  atmospheric  oxygen  makes  the  life  processes  of  most 
bacteria  more  easy,  but  is  not  indispensable  when  available  substances 
are  present  which  can  be  broken  up  with  sufficient  ease.  The  standard 
of  availability  is  verv  different  for  different  bacteria. 


88  PRINCIPLES  OF  BACTERIOLOGY 

In  the  presence  of  oxygen  some  of  the  decomposition  products  that  are 
formed  by  the  attack  of  the  anaerobic  bacteria  are  further  decomposed 
and  oxidized  by  the  aerobes;  they  are  thereby  rendered,  as  a  rule,  inert 
and  consequently  harmless.  Some  bacteria  have  adapted  themselves 
to  the  exclusive  use  of  compound  oxygen,  using  those  compounds  from 
which  oxygen  can  be  obtained,  and  others — the  obligatory  anaerobes 
— are  able  to  live  only  in  the  presence  of  free  oxygen.  The  facts  of 
anaerobiosis  are  of  great  importance  to  technical  biology  and  to  path- 
ology. Many  parasitic  bacteria  are  found  to  produce  far  more  poison 
in  the  absence  of  air  than  in  its  presence.  The  following  three  types 
of  chemical  activity  can  be  separated:  1.  The  bacteria  develop  their 
tissues.  2.  The  bacteria  produce  and  liberate  ferments  or  enzymes 
which  tend  to  make  the  foodstuff  in  their  neighborhood  more  assimi- 
lable. 3.  The  bacteria  assimilate  substance  and  liberate  it  changed  to 
other  material.  These  changes  may  be  due  to  ferments  retained  in 
the  cells. 

Fermentation. — The  term  fermentation  is  differently  used  by  different 
authors.  Some  call  every  kind  of  decomposition  due  to  bacteria  or 
their  products  a  fermentation,  speaking  thus  of  the  putrefactive  fermen- 
tation of  albuminous  substances;  others  limit  the  term  to  the  process 
when  accompanied  by  the  visible  production  of  gas;  others,  again, 
take  fermentation  to  mean  only  the  decomposition  of  carbohydrates, 
with  or  without  gas-production. 

Fermentation  may  be  defined  as  a  chemical  decomposition  of  an 
organic  compound,  induced  by  the  life  processes  of  living  organisms 
(organized  ferments),  or  by  chemical  substances  thrown  off  from  the 
bacteria  (unorganized  or  chemical  ferments  or  enzymes).  In  the  first 
the  action  is  due  to  the  life  processes  necessary  for  the  growth  of  the 
organisms  producing  the  ferment,  as  in  the  formation  of  acetic  acid 
from  alcohol  by  the  action  of  the  vinegar  plant,  and  in  the  second  the 
enzyme,  either  within  or  outside  of  the  organism  and  having  no 
direct  connection  with  the  growth  of  the  organism,  causes  a  structural 
change  without  losing  its  identity,  as  in  digestion.  E.  Buchner 
(Berichte  d.  Deutsch.  chem.  Gesellsch.,  xxx.  117-124  and  1110-1113) 
has  shown  that,  even  in  those  cases  of  fermentation  in  which  formerly 
it  was  believed  the  organized  cell  itself  was  necessarily  concerned,  the 
cell  protoplasm  squeezed  from  its  capsule  is  able  to  cause  the  same 
changes  as  the  organized  cells.  This  brings  fermentation  by  unorgan- 
ized and  organized  ferments  very  closely  together,  the  one  being  a  sub- 
stance thrown  off  from  the  cell,  the  other  a  substance  ordinarily  retained 
in  the  cell.  The  increase  of  both  ceases  with  the  death  of  the  bacteria 
producing  them.  These  enzymes,  even  when  present  in  the  most 
minute  quantities,  have  the  power  of  splitting  up  or  decomposing 
complex  organic  compounds  into  simpler,  more  easily  soluble  and 
diffusible  molecules.  The  changes  thus  made  may  greatly  aid  in 
rendering  the  foodstuff  suitable  for  bacterial  growth.  We  can  only 
speak  of  chemical  ferments  when  it  can  be  demonstrated  that  the 
fermentation  continues  in  the  absence  of  all  living  bacteria.  This 


VITAL  PHE\".M1-:XA  OF  BACTERIA  89 

may  be  accomplished  by  the  addition  of  disinfectants — carbolic   acid, 
chloroform,   ether,   etc. — to  the  cultures  or  by  filtration. 

(  HAH.U  TKRISTICS  OF  FEKMKXTs. — Ferments  are  non-dialyzable. 
They  withstand  moderate  dry  heat,  but  are  usually  destroyed  in 
watery  solutions  by  a  temperature  of  over  70°  C.  They  are  injured 
by  acids,  especially  the  inorganic  ones,  but  are  resistant  to  all  alkalies. 
A  simple  example  of  bacterial  fermentation  of  carbohydrates  produced 
by  an  enzyme  is  that  of  grape-sugar: 

C6H1206  2C2H60  +  2CO2 

Grape-sugar.  2  Alcohol.  2  Carbon  dioxide. 

Or, 

C6H1206  2C3H60S 

Grape-sugar.  2  Lactic  acid. 


gj2g  ->42 

Grape-sugar.  3  Acetic  acid. 

Far  less  common  is  oxidizing  fermentation,  as  in  the  production  of 
acetic  acid  from  alcohol.  Here  the  energy  is  acquired  not  by  the 
decomposition  but  by  the  oxidation  of  the  alcohol. 

The  proteolytic  or  peptonizing  ferments  which  are  somewhat  anal- 
ogous to  trypsin — being  capable  of  changing  albuminous  bodies  into 
soluble  and  diffusible  substances — are  very  widely  distributed.  The 
liquefaction  of  gelatin,  which  is  chemically  allied  to  albumin,  is  due  to 
the  presence  of  a  proteolytic  ferment  or  trypsin.  The  production  of  pro- 
teolytic ferments  by  different  cultures  of  the  same  varieties  of  bacteria 
varies  considerably — far  more  than  is  generally  supposed.  Even  among 
the  freely  liquefying  bacteria,  such  as  the  cholera  spirillum  and  the 
staphylococcus,  poorly  liquefying  varieties  have  been  repeatedly  found. 
These  observations  have  detracted  considerably  from  the  value  in  cul- 
tures of  the  property  of  liquefying  gelatin  as  a  positive  diagnostic  char- 
acteristic. Most  conditions  which  are  unfavorable  to  the  growth  of 
bacteria  seem  to  interfere  also  with  their  liquefying  power. 

Certain  bitter-tasting  products  of  decomposition  are  formed  by 
liquefying  bacteria  in  media  containing  proteid,  as,  for  example,  in  milk. 

Diastatic  ferments  convert  starch  into  sugar.  That  these  are  pro- 
duced by  bacteria  is  shown  by  mixing  starch  paste  with  cultures  to  the 
resulting  mixture  of  which  thymol  has  been  added,  and  keeping  the 
digestion  for  six  to  eight  hours  in  the  incubating  oven;  then,  on  the 
addition  of  Fehling's  solution  and  heating,  the  reaction  for  sugar  appears 
— the  reddish-yellow  precipitate  due  to  the  reduction  of  the  copper. 
Bacteria  may  be  directly  tested  for  sugar  also  by  boiling  potato-broth 
cultures  and  using  the  extract. 

Inverting  ferments  (that  is,  those  which  convert  polysaccharides  into 
monosaccharides)  are  of  very  frequent  occurrence.  Bacterial  invertin 
withstands  a  temperature  of  100°  C.  for  more  than  an  hour,  and  is 
produced  in  culture  media  free  from  proteid.  For  more  details  as  to  the 
action  of  ferments  on  sugars  see  chapter  on  the  colon-typhoid  groups. 


90  PRINCIPLES  £>F  BACTERIOLOGY 

Rennin-like  ferments  (substances  having  the  power  of  coagulating  milk 
with  neutral  reaction,  independent  of  acids)  are  found  not  infrequently 
among  bacteria.  The  B.  prodigiosus,  for  instance,  in  from  one  to  two 
days  coagulates  to  a  solid  mass  milk  which  has  been  sterilized  at  55° 
to  60°  C.  These  ferments  have  not  been  thoroughly  investigated ;  they 
are  probably  present,  however,  in  all  species  of  bacteria  which  coagu- 
late milk,  even  though  the  organisms  also  produce  acid. 

Fermentation  yields  products  that  are  poisonous  to  the  ferment; 
hence  fermentation  ceases  when  the  nutriment  is  exhausted  or  the  fer- 
mentation is  in  excess.  Different  kinds  of  fermentation  obtain  specific 
names,  according  to  the  products.  Thus  acetic,  yielding  acetic  acid;  alco- 
holic or  vinous,  yielding  alcohol;  ammoniacal,  yielding  ammonia;  amylic, 
yielding  amylic  alcohol;  benzole,  yielding  benzoic  acid;  butyric,  yielding 
butyric  acid;  lactic,  yielding  lactic  acid;  and  viscous,  yielding  a  gummy 
mass. 

Pigment  Production. — Pigments  have  no  known  importance  in  con- 
nection -with  disease,  but  are  of  interest  and  have  value  in  identifying 
bacteria.  Their  chemical  composition  is  not  generally  known. 

RED  AND  YELLOW  PIGMENTS. — Of  the  twenty-seven  red  and  yellow 
bacteria  studied  by  Schneider,  almost  all  produce  pigments  soluble  in 
alcohol  and  insoluble  in  water.  The  larger  majority  of  these  possess 
in  common  the  property  of  being  colored  blue-green  by  sulphuric  acid 
and  red  or  orange  by  a  solution  of  potash.  Though  varying  consider- 
ably in  their  chemical  composition  and  in  their  spectra,  they  may  be 
classified,  for  the  most  part,  among  that  large  group  of  pigments  com- 
mon to  both  the  animal  and  vegetable  kingdoms  known  as  lipochromes, 
and  to  which  belong  the  pigments  of  fat,  yolk  of  egg,  the  carotin  of 
carrots,  turnips,  etc. 

VIOLET  PIGMENTS. — Certain  bacteria  produce  violet  pigments,  also 
insoluble  in  water  and  soluble  in  alcohol,  but  insoluble  in  ether,  benzol, 
and  chloroform.  These  are  colored  yellow  when  treated  in  a  dry  state 
with  sulphuric  acid  and  emerald-green  with  potash  solution. 

BLUE  PIGMENTS,  such  as  the  blue  pyocyanin,  are  also  produced  by 
the  so-called  fluorescent  bacteria,  along  with  a  pigment  named  bac- 
teriofluorescin.  In  cultures  the  fluorescence  is  at  first  blue;  later,  as 
the  cultures  become  alkaline  it  is  green. 

Numerous  investigations  have  been  made  to  determine  the  cause  of 
the  variation  in  the  chromogenic  function  of  bacteria.  All  conditions 
which  are  unfavorable  to  the  growth  of  the  bacteria  decrease  the  pro- 
duction of  pigment,  as  cultivation  in  unsuitable  media  or  at  too  low 
or  too  high  a  temperature,  etc.  The  B.  prodigiosus  produces  no 
pigndent  at  37°  C.,  and  when  transplanted  at  this  temperature,  even 
into  favorable  media,  the  power  of  pigment  production  is  gradually  lost. 
B.  pyocyaneus  does  not  produce  pigment  under  anaerobic  conditions. 

Ordinarily  colorless  species  of  bacteria  sometimes  produce  pigments. 
Thus  yellow  colonies  of  the  pneumococcus  have  been  observed,  and 
colored  varieties  of  the  streptococcus  pyogenes.  Occasionally  colored 
and  uncolored  colonies  of  the  same  species  of  bacteria  may  be  seen  to 


fi    UNIVERSITY  1 
VITAL  PHE^^mCfpTERIA  91 


occur  side  by  side  in  one  plate  culture,  as,  for  example,  the  staphylo- 
coccus  pyogenes. 

Alkaline  Products  and  the  Fermentation  of  Urea. — Aerobic  bacteria 
always  produce  alkaline  products  from  albuminous  substances  in  cul- 
ture media  free  from  sugar.  Many  species  of  bacteria  produce  acids 
in  the  presence  of  sugars,  which  explains  the  fact  that  neutral  or  slightly 
alkaline  broth  often  becomes  acid  at  first  from  the  fermentation  of  the 
sugar  contained  in  the  meat  used  for  making  the  media.  When  the 
sugar  is  used  up  the  reaction  often  becomes  alkaline,  as  the  production 
of  alkalies  continues.  The  substances  producing  the  alkalinity  in  cul- 
tures are  chiefly  ammonia,  amine,  and  the  ammonium  bases. 

The  conversion  of  urea  into  carbonate  of  ammonia  affords  an 
example  of  the  production  of  alkaline  substances  by  bacteria: 

CO(NH,)2  +  2H20  00S(XH4), 

Urea.  2  Water.  Ammonium  carbonate. 

The  power  of  decomposing  urea  is  not  widespread  among  bacteria. 
Of  sixty  species  investigated  by  Lehman,  three  only  developed  the  odor 
of  ammonia  from  sterilized  human  urine. 

Ptomains. — Nencki,  and  then  later  Brieger,  Vaughan,  and  others,  suc- 
ceeded in  preparing  organic  bases  of  a  definite  chemical  composition  out 
of  putrefying  fluids — meat,  fish,  old  cheese,  and  milk — as  well  as  from 
pure  bacterial  cultures.  Some  of  these  were  found  to  exert  a  poisonous 
effect,  while  others  were  harmless.  The  poisons  may  be  present  in 
the  decomposing  cadaver  (hence  the  name  ptomain,  from  --wfjia., 
putrefaction),  and,  in  consequence,  have  to  be  taken  into  considera- 
tion in  questions  of  legal  medicine.  They  may  be  formed  also  in  the 
living  human  body,  and,  if  not  made  harmless  by  oxidation,  may  come 
to  act  therein  as  self-poisons  or  leucomains.  They  are  now  known 
not  to  be  the  substances  which  excite  the  specific  poisonous  effects  of 
bacteria.  The  latter  are  easily  destroyed  by  heat,  and  have  entirely 
different  characteristics. 

Many  ptomains  are  known  already  and  the  elementary  composition 
of  each  made  out,  and  among  them  are  some  whose  exact  chemical  con- 
stitution is  established.  Especially  interesting  is  the  substance  cada- 
verin,  which  was  separated  by  Brieger  from  portions  of  decomposing 
dead  bodies  and  from  cholera  cultures,  by  reason  of  the  fact  that 
Ladenburg  prepared  it  synthetically  and  showed  it  to  be  pentame- 
thylenediamin  [(NH2)2(CH2)5].  The  cholin  group  is  particularly  inter- 
esting. Cholin  itself  (C5H,5NO2)  arises  from  the  hydrolytic  breaking-up 
of  lecithin,  the  fat-like  substance  found  in  the  brain  and  other  nervous 
tissue.  By  the  oxidation  of  cholin  there  can  be  produced  the  highly 
toxic  muscarin,  found  by  Schmiedeberg  in  a  poisonous  toadstool  and 
by  Brieger  in  certain  decomposing  substances: 

C5H15N02        +        O        =        C5H15N03 
Cholin.  Muscarin. 

The  ptomain  tyrotoxicon  was  obtained  from  cheese,  milk,  and  ice- 
cream by  Vaughan. 


92  PRINCIPLES  OF  BACTERIOLOGY 

Pyocyanin  (C14H14N2O),  which  produces  the  color  of  blue  or  blue- 
green  pus,  is  a  ptomainic  pigment.  Similar  bodies  of  a  basic  nature 
may  be  found  in  the  intestinal  contents  as  the  products  of  bacterial 
decomposition.  Some  of  these  are  poisons  and  can  be  absorbed  into 
the  body.  Some  believe  the  symptoms  designated  as  coma  and  tetany 
may  be  ascribed  to  the  absorption  of  substances  of  this  nature.  Since 
the  name  ptomain  was  given  to  the  poisonous  products  of  bacterial 
growth  before  these  products  were  chemically  understood  it  is  by  many 
wrongly  applied  to  all  poisons  found  in  food,  as  in  cases  of  poisoning 
due  to  decomposing  meat  or  sausage  or  to  cheese  or  milk.  These  are 
sometimes  true  toxins  or  even  living  bacteria. 

The  isolation  of  these  substances  can  here  be  only  briefly  referred  to. 
According  to  Brieger's  method,  which  is  the  one  now  generally  em- 
ployed, the  cultures  having  a  slight  acid  reaction  (HC1)  are  boiled  down, 
filtered,  and  the  filtrate  concentrated  to  a  syrupy  consistency,  dissolved 
in  96  per  cent,  alcohol,  purified  and  precipitated  by  means  of  an  alco- 
holic solution  of  bichloride  of  mercurv. 


THE  MORE   COMPLICATED  PROTEID  POISONS. 

These  are  divided  into  bacterial  proteins,  toxins,  and  endotoxins. 

Bacterial  Proteins. — These  substances  are  bacterial  poisons  which  are 
little  or  not  at  all  specific,  that  excite  fever,  inflammation,  and  suppu- 
ration, and  resist  the  boiling  temperature.  They  are  usually  extracted 
by  boiling  cultures  in  0.5  per  cent,  sodium  hydrate  solution.  The  pro- 
teins are  precipitated  in  weak  acid  solutions.  Tuberculin  is  the  most 
important  of  the  group;  mallein  is  another. 

Like  mallein,  according  to  Buchner  and  Romer,  all  bacterial  proteins 
are  very  similar  in  their  action,  and  Matthes  maintains  that  deutero- 
albumose,  which  is  obtained  by  the  action  of  pepsin  on  albumin  and 
has  no  connection  with  bacteria,  has  an  effect  on  tuberculous  guinea- 
pigs  somewhat  similar  to  tuberculin. 

Toxins. — Toxins  are  poisonous  synthetical  products  of  bacterial 
growth. 

The  exact  composition  of  toxins  has  not  as  yet  been  discovered,  but 
it  is  believed  that  they  are  of  proteid  character.  At  first  all  the  toxins 
were  supposed  to  be  albumins,  but  recently  some  of  the  most  important, 
such  as  those  produced  by  the  tetanus  and  diphtheria  bacilli,  have  been 
shown  to  possess  characteristics  which  separate  them  from  that  class. 
Toxins  are  formed  during  the  growth  of  bacteria  in  media  containing 
no  proteid,  but  more  abundantly  when  it  is  present.  Toxins  are  divided 
into  extracellular  and  intracellular  poisons.  Thus,  the  toxins  produced 
by  the  diphtheria  and  tetanus  bacilli  during  their  growth  in  the  tissues 
or  culture  media  are  largely  given  up  to  the  culture  fluid,  but  little 
remaining  in  the  bacterial  protoplasm,  while  the  toxins  elaborated  by 
the  typhoid,  tubercle,  glanders,  and  colon  bacilli,  and  indeed  by  the 
majority  of  parasitic  and  saprophytic  bacteria,  are  largely  retained  in 
the  bodies  of  the  bacteria  until  their  death  and  destruction. 


VITAL  PHENOMENA  <>F  BACTERIA  93 

Among  the  properties  of  the  extracellular  toxins  are  the  following: 
They  are,  so  far  as  known,  uncrvstallizable,  and  thus  differ  fromptomains; 
they  are  soluble  in  water  and  they  are  dialyzable;  they  are  precipitated 
along  with  proteids  by  concentrated  alcohol,  and  also  by  ammonium 
sulphate;  if  they  are  proteids  they  are  either  albumoses  or  allied  to  the 
albumoses;  they  are  relatively  unstable,  having  their  toxicity  dimin- 
ished or  destroyed  by  heat  (the  degree  of  heat,  etc.,  which  is  destructive 
varies  much  in  different  cases).  Their  potency  is  often  altered  in  the 
precipitations  practised  to  obtain  them  in  a  pure  or  concentrated  condi- 
tion, but  among  the  precipitants  ammonium  sulphate  has  little  if  any 
harmful  effect.  Regarding  the  intracellular  toxins  which  are  more 
intimately  associated  with  the  bacterial  cell  we  know  much  less,  but 
it  is  probable  that  their  nature  is  similar,  though  some  of  them  at  least 
are  not  so  easily  injured  by  heat — e.  g.,  in  the  case  of  the  product 
of  tubercle  bacilli.  In  the  case  of  all  toxins  the  fatal  dose  for  an 
animal  varies  directly  with  the  species,  body  weight,  age,  and  previous 
conditions  as  to,  e.  g.,  food,  temperature,  etc.  In  estimating  the 
minimal  lethal  dose  of  a  toxin  these  factors  must  be  carefully  con- 
sidered. 

The  following  is  the  method  usually  employed  for  obtaining  concen- 
trated extracellular  toxins.  The  toxic  fluid  is  placed  in  a  shallow  dish, 
and  ammonium  sulphate  crystals  are  well  stirred  in  till  no  more  dis- 
solve. Fresh  crystals  to  form  a  bulk  nearly  equal  to  that  of  the  whole 
fluid  are  added,  and  the  dish  is  set  in  an  incubator  at  37°  C.  (98.6°  F.) 
overnight.  Next  day  a  brown  scum  of  precipitate  will  be  found  floating 
on  the  surface.  This  contains  the  toxin.  It  is  skimmed  off  with  a 
spoon,  placed  in  watch-glasses,  and  these  are  dried  in  vacuo  and  stored 
in  the  dark,  also  in  vacuo,  or  in  an  exsiccator  containing  strong  sul- 
phuric acid.  For  use,  the  contents  of  one  are  dissolved  in  a  little 
normal  saline  solution. 

The  comparison  of  the  action  of  bacteria  in  the  tissues  in  the  pro- 
duction of  these  toxins  to  what  takes  place  in  the  gastric  digestion,  has 
raised  the  question  of  the  possibility  of  the  elaboration  by  these  bac- 
teria of  ferments,  by  which  the  process  may  be  started.  It  would  not 
be  prudent  to  dogmatize  as  to  whether  the  toxins  do  or  do  not  belong 
to  such  an  ill-defined  group  of  substances  as  the  ferments.  It  may  be 
pointed  out,  however,  that  the  essential  concept  of  a  ferment  is  that  of 
a  body  wrhich  can  originate  change  without  itself  being  changed,  and 
no  evidence  has  been  adduced  that  toxins  fulfil  this  condition.  Another 
property  of  ferments  is  that,  so  long  as  the  products  of  fermentation 
are  removed,  the  action  of  a  given  amount  of  ferment  is  indefinite. 
Again,  in  the  case  of  toxins  no  evidence  of  such  an  occurrence  has  been 
found.  A  certain  amount  of  a  toxin  is  always  associated  with  a  given 
amount  of  disease  effect,  though  a  process  of  elimination  of  waste 
products  must  be  all  the  time  going  on  in  the  animal's  body.  Again,  too 
much  importance  must  not  be  attached  to  loss  of  toxicity  by  toxins  at 
relatively  low  temperatures.  Many  proteids  show  a  tendency  to  change 
at  such  temperatures;  for  instance,  if  egg  albumen  be  kept  long  enough 


94  PRINCIPLES  OF  BACTERIOLOGY 

at  55°  C.  nearly  the  whole  of  it  will  be  coagulated.  Such  considerations 
suggest  that  the  relation  of  toxic  action  to  fermentation  must  be  left  an 
open  question. 

SIMILAR  VEGETABLE  AND  ANIMAL  POISONS. — Within  recent  years 
it  has  been  found  that  the  bacterial  poisons  belong  to  a  group  of  toxic 
bodies  all  presenting  very  similar  properties;  other  members  of  which 
occur  widely  in  the  vegetable  and  animal  kingdoms.  Among  plants 
the  best-known  examples  are  the  ricin  and  abrin  poisons,  obtained  by 
making  water  emulsions  of  the  seeds  of  the  Ricinus  communis  and  the 
Abrus  precatorius  (jequirity),  respectively.  The  chemical  reactions  of 
ricin  and  abrin  correspond  to  those  of  the  bacterial  toxins.  They  are 
soluble  in  water;  they  are  precipitable  in  alcohol,  but  being  less  easily 
dialyzable  than  the  albumoses  they  have  been  called  toxalbumins. 
Their  toxicity  is  seriously  impaired  by  boiling,  and  they  also  gradually 
become  less  toxic  on  being  kept.  Both  are  among  the  most  powerful 
poisons  known,  ricin  being  the  more  fatal. 

It  is  also  certain  that  the  poisons  of  scorpions  and  of  poisonous  snakes 
belong  to  the  same  group.  The  poisons  derived  from  the  latter  are 
usually  called  venins,  and  a  very  representative  group  of  such  venins 
derived  from  different  species  has  been  studied.  To  speak  generally, 
there  is  derivable  from  the  natural  secretions  of  the  poison  glands  a 
series  of  venins  which  have  all  the  reactions  of  the  bodies  previously 
considered.  Like  ricin  and  abrin,  they  are  not  so  easily  dialyzable  as 
bacterial  toxins,  and  therefore  they  have  also  been  classed  as  toxalbu- 
mins. While  up  to  the  present  we  have  not  been  able  to  discover  the 
exact  chemical  composition  of  any  toxin,  or  even  to  obtain  it  in  a  pure 
state,  many  interesting  facts  upon  the  nature  of  toxins  have  been  dis- 
covered by  physiological  methods. 

Ehrlich's  Theories  as  to  the  Nature  of  Extracellular  Toxins. — From  a 
large  number  of  most  carefully  conducted  experiments  with  the  toxin 
and  antitoxin  of  diphtheria,  Ehrlich  has  formulated  a  theory  con- 
cerning the  constitution  of  the  former.  This  theory  has  undergone 
several  modifications  since  it  was  first  proposed,  and  it  is  difficult  to 
give  an  exact  statement  of  it  as  it  now  stands.  However,  we  will  attempt 
to  state  in  condensed  form  its  essential  points  as  follows: 

Toxins  and  antitoxins  neutralize  one  another  after  the  manner  of 
chemical  reagents.  The  chief  reasons  for  this  belief  lie  in  the  observed 
facts:  (a)  that  neutralization  takes  place  more  rapidly  in  concentrated 
than  in  dilute  solutions,  and  (6)  that  warmth  hastens  and  cold  retards 
neutralization.  From  these  observations  Ehrlich  concludes  that  toxins 
and  antitoxins  act  as  chemical  reagents  do  in  the  formation  of  double 
salts.  A  molecule  of  the  poison  requires  an  exact  and  constant  quan- 
tity of  the  antitoxin  in  order  to  produce  a  neutral  or  harmless  substance. 
This  implies  that  a  specific  atomic  group  in  the  toxin  molecule  com- 
bines with  a  certain  atomic  group  in  the  antitoxin  molecule. 

The  toxins,  however,  are  not  simple  bodies,  but  easily  split  into  other 
substances  which  differ  from  one  another  in  the  avidity  with  which  they 
combine  with  antitoxin. 


VITAL  /V//-:.\VM//-:.Y.I  or  HACTERIA  95 

These  derivatives  Ehrlich  calls  prototoxins,  deuterotoxins,  and  tri- 
totoxins. 

All  forms  of  toxins  are  supposed  to  consist  of  two  modifications, 
which  combine  in  an  equally  energetic  manner  with  antitoxin  or  with 
suitable  receptors  in  the  cells,  but  differ  in  their  resistance  to  heat  and 
other  destructive  agents. 

The  less-resistant  form  passes  readily  into  a  toxoid  substance  which 
has  the  same  affinity  for  the  antitoxin  or  the  cell  receptors  as  the  original 
toxin,  but  is  not  poisonous.  The  facts  observed,  Ehrlich  thinks,  are 
best  explained  on  the  supposition  that  the  toxic  molecule  contains  two 
independent  groups  of  atoms,  one  of  which  may  be  designated  as  the 
haptophorous  and  the  other  as  the  toxophorous  group.  It  is  by  the 
action  of  the  haptophorous  group  that  toxin  unites  with  antitoxin  or 
the  sensitive  cell  molecule. 

The  toxophorous  group  is  unstable,  but  after  its  destruction  the 
molecule  still  unites  with  the  antitoxin  or  the  sensitive  molecule  through 
its  retained  haptophorous  group. 

Specific  antitoxins  can  be  produced  not  only  with  toxins,  but  with 
toxoids. 

Bordet  believes,  in  contradistinction  to  Ehrlich,  that  toxin  unites  in 
different  multiples  with  antitoxin,  so  that  the  toxin  molecule  may  have 
its  affinity  slightly,  partly,  or  wholly  satisfied  by  antitoxin.  Slightly 
satisfied,  it  is  still  feebly  toxic;  combined  with  a  larger  amount  of  anti- 
toxin, it  is  not  toxic;  but  still  may,  when  absorbed  into  the  system,  lead 
to  the  production  of  antitoxin.  Fully  saturated,  it  has  no  poisonous 
properties  and  no  ability  to  stimulate  the  production  of  antitoxin. 

The  most  important  of  the  extracellular  toxins  are  those  produced 
by  the  diphtheria  and  tetanus  bacilli.  These  are  very  powerful; 
0.0000001  gram  of  the  dried  filtrate  of  a  tetanus  culture  will  frequently 
kill  a  white  mouse,  while  one-tenth  of  that  amount  of  dried  diphtheria 
filtrate  has  killed  a  guinea-pig. 

The  same  bacterium  may  produce  several  entirely  distinct  toxins, 
thus,  according  to  Madsen  and  Ehrlich,  the  specific  tetanus  poison  con- 
sists of  twTo  toxins,  tetanospasmin  and  tetanolysin.  To  the  first  of  these 
the  tetanic  convulsions  are  due,  while  the  second  has  a  hsemolytic 
action. 

When  the  tetanus  toxins  are  placed  in  the  blood  tetanolysin  largely 
combines  with  the  blood  corpuscles,  while  the  tetanospasmin  combines 
with  the  nerve  cells.  Each  of  these  substances  produces  in  animals  a 
specific  antitoxin.  To  obtain  diphtheria  and  tetanus  toxins  for  injection 
in  animals  the  bacilli  are  grown  in  slightly  alkaline  beef-broth  for  from 
seven  to  ten  days.  The  broth  is  then  filtered  and  preserved.  Tetanus 
toxin  is  produced  under  anaerobic  conditions;  diphtheria,  under  free 
access  of  oxygen.  (See  special  chapters  on  these  bacteria.) 

Bacterial  Endotoxins  or  Proteids. — The  bacterial  poisons  which  reside 
in  the  bodies  of  the  bacteria  are  mostly  yielded  up  only  after  the  death 
of  the  organisms.  Here,  in  the  invaded  animal,  the  disease  effects 
are  more  closely  associated  with  the  actual  presence  of  the  bacteria 


96  PRINCIPLES  OF  BACTERIOLOGY 

in  the  vicinity  than  in  the  case  of  the  extracellular  toxins.  These  sub- 
stances are  extracted  by  the  method  proposed  by  Koch  and  Buchner, 
of  first  crushing  the  bacteria  in  a  moist  or  dried  condition,  and  then  of 
obtaining  their  contents  with  the  aid  partly  of  the  hydraulic  press. 
In  this  way  a  large  series  of  impure  bacterial  proteids  was  obtained, 
which,  though  differing  in  some  respects,  exhibited  mainly  the  same 
properties. 

Altogether  different  from  these  poison  effects  are  the  immunization 
processes  produced  by  the  cell  substances  of  bacteria,  whether  they  be 
obtained  from  bacterial  bodies  or  from  chemical  preparations.  These 
processes  have  nothing  to  do  with  the  toxic  action  of  the  cell  proteids, 
but  rather  depend  upon  the  introduction  of  suitable  receptors  which 
give  rise  to  the  bactericidal  protective  powers — lysin,  precipitin,  and 
agglutinin. 

For  the  present  we  may  assume  with  certainty  that  such  receptors 
exist  only  in  the  unchanged  bacterial  cells,  which,  like  cholera  vibrio, 
pneumococcus,  etc.,  give  up  in  toto  their  destructive  processes;  on  the 
other  hand,  we  may  say  pretty  surely  that  forcible  extraction — that  is, 
production  of  chemical  proteid  preparation — so  changes  most  of  the 
atomical  grouping  that  little  or  no  bactericidal  reaction  results  from 
their  introduction,  and  that  these  albuminous  substances  produce  only 
the  same  reaction  as  other  outside  albuminous  substances — i.  e.,  the 
formation  of  a  specific  precipitin,  which,  however,  is  closely  allied  to 
agglutinin.  It  is  very  probable,  on  the  contrary,  that  in  the  substance 
so  carefully  prepared  as  Koch's  tuberculin  and  Buchner's  plasmin,  from 
the  tubercle  bacillus  and  the  cholera  vibrio,  the  specific  receptors  may 
be  retained,  so  that  these  preparations  produce  bactericidal  arid  immuni- 
zation processes. 

SUMMARY. — 1.  One  group  of  bacteria  produces  as  free  secretions 
true  toxins.  After  extraction  of  this  soluble  poison  there  remains  a  pure 
unspecific  bacterial  residue.  Type:  diphtheria. 

2.  Another   large  group  possesses   apparently  only  endotoxins,  true 
toxins  which  are  more  or  less  closely  bound  to  the  living  cell,  and  which 
are  only  in  a  small  degree  separable  in  unchanged  condition  perhaps 
outside  of  the  body.     On  death  of  the  cell  they  become  partly  free, 
partly  remain  united,  or  become   secondary    poisonous   modifications 
no  longer  of  the  nature  of  toxins.     In  this  group,  therefore,  the  dead 
cell  bodies  cannot  be  entirely  freed  without  residue  from  the  poisons; 
the  pure  proteid  cannot  be  clearly  identified  by  its  individual  action,  i 
With  this  reservation,  however,  the  proteid  action  can  be  demonstrated. 
Type:   cholera,  typhoid,  pneumococcus. 

3.  A  third  group  yields  perhaps  no  true  toxins,  not  even  intraplas- 
matically.     The  cell  plasma  contains  poisons  of  another  kind  which 
obscures    the    typical    proteid    action.      Type:    anthrax,    tuberculosis. 
Possibly  by  further  investigation  Groups  2  and  3  may  be  united. 

The  pyogenic  action  of    their   proteids   is   common  to  all   bacteria,  i 
this  depending   principally   upon   their   being   extraneous  albuminous 
substances.     Pyogenic    effects   may  be   produced  in  like   manner    by 


VITAL  PHEXOMEXA  OF  BACTERIA  97 

extraneous  albumins  of  non-bacterial  origin.  That  every  extraneous 
al bun li nous  substance  is  harmful  to  the  organism  which  seeks  to  resist 
it>  action  is  shown  by  those  specific  precipitating  ferments,  the  pre- 
cipitins,  which  are  produced  in  the  organisms  after  the  introduction 
of  every  extraneous  albumin. 

Sulphuretted  Hydrogen.— Sulphuretted  hydrogen  is  a  very  common 
bacterial  product.  Its  presence  is  determined  by  pasting  a  piece  of  paper 
moistened  with  lead  acetate  inside  the  neck  of  the  flask  containing  the 
culture,  closing  the  mouth  with  a  cotton-wool  stopper,  and  over  this 
again  an  India-rubber  cap  (black  rubber  free  from  sulphur).  The 
paper  is  colored  at  first  brownish  and  later  black;  repeated  observation 
is  necessary,  as  the  color  sometimes  disappears  toward  the  end  of  the 
reaction.  Apparently  negative  results  should  not  be  rashly  accepted 
as  conclusive. 

Sulphuretted  hydrogen  may  be  formed: 

1.  From  albuminous  substances.     This  power,   according  to  Petri 
and  Maassen,  of  forming  sulphuretted  hydrogen,  particularly  in  liquid 
culture  media  containing  much  peptone  (5  to  10  per  cent.)  is    pos- 
sessed, though  in  different  degree,  by  all  bacteria  examined  by  them; 
only  a  few  bacteria  form  H2S  in  bouillon  in  the  absence  of  peptone, 
while  about  50  per  cent,  in  media  containing  1  per  cent,  peptone. 

2.  From  powdered  sulphur.     All  bacteria  produce  in  culture  media 
to  which  pure  powdered  sulphur  is  added  considerably  more  H2S  than 
without  this  addition.     Petri  and  Maassen  suggest  that  this  is  due  to 
the  nascent  hydrogen  produced  by  the  bacteria. 

3.  From  thiosulphates  and  sulphites.     Studied  particularly  in  yeast, 
but  demonstrated  also  by  Petri  and  Maassen  in  several  bacteria. 

The  presence  of  sugar  in  the  culture  does  not  affect  the  production 
of  H2S  by  bacteria,  but  saltpetre  reduces  it,  nitrites  being  formed.  The 
absence  of  oxygen  favors  the  production  of  H2S.  Light  diminishes 
the  development  of  H2S  by  facultative  anaerobes,  sulphates  being 
formed  instead. 

Reduction  Processes. — All  bacteria,  as  we  have  seen,  possess  the 
property  of  converting  sulphur  into  sulphuretted  hydrogen,  for  which 
purpose  is  required  the  presence  of  nascent  hydrogen.  The  following 
processes  depend  also  in  part  upon  the  action  of  nascent  hydrogen: 

1.  The   reduction    of   blue   litmus    pigments,    methylene  blue,  and 
indigo  to  colorless  substances.    The  superficial  layer  of  cultures  in  con- 
tact with  the  air  shows  often  no  reduction,  only  the  deeper  layers  being 
affected.    By  agitation  with  access  of  air  the  colors  may  be  again  restored, 
but,  at  the  same  time,  if  acid  has  been  formed,  the  litmus  pigment  is 
turned   red.     According   to    Cohn,  the   property   of    reducing    litmus 
belongs  to  all  liquefying  bacteria,  but  some  non-liquefying  species  also 
possess  it. 

2.  The  reduction  of  nitrates  to  nitrites  and  ammonia.     The  first  of 
these  properties  seems  to  pertain  to  a  great  many  bacteria — at  least 
Petri  and  Maassen  found  in  six  species,  grown  in  bouillon  containing 
2.5  to  5  per  cent,  peptone  and  0.5  per  cent,  nitrate,  that  almost  all  pro- 


98  PRINCIPLES  OF  BACTERIOLOGY 

duced  nitrite  abundantly;  once  only  was  ammonia  observed.  In  a 
number  of  bacteria  studied  by  Rubner  only  one  failed  to  produce  nitrite. 
The  test  for  nitrites  is  made  as  follows :  Two  bouillon  tubes  containing 
nitrates  are  inoculated,  and,  along  with  two  uninoculated  tubes,  are 
allowed  to  remain  in  the  incubator  for  several  days;  then  to  the  cultures 
and  control  test  is  added  a  small  quantity  of  colorless  iodide  of  starch 
solution  (thin  starch  paste  containing  0.5  per  cent,  potassium  iodide) 
and  a  few  drops  of  pure  sulphuric  acid.  The  control  tubes  remain 
colorless  or  become  gradually  slightly  blue,  while  if  nitrites  are  present 
a  dark-blue  or  brown-red  coloration  is  produced. 

The  demonstration  of  ammonia  is  made  by  the  addition  of  Nessler's 
reagent  to  culture  media  free  from  sugar.  In  bouillon,  if  ammonia  be 
present,  Nessler's  reagent  is  almost  immediately  reduced  to  black 
mercurous  oxide.  A  strip  of  paper  saturated  with  the  reagent  can  also 
be  suspended  over  the  bouillon  tube,  or  this  can  be  distilled  at  a  low 
temperature  with  the  addition  of  magnesium  oxide  and  the  distillate 
treated  with  Nessler's  reagent.  A  yellow  to  red  coloration  indicates  the 
presence  of  ammonia.  Controls  are  necessary. 

Aromatic  Products  of  Decomposition. — Many  bacteria  produce  aromatic 
substances  as  the  result  of  their  growth.  The  best  known  of  these  are 
indol,  skatol,  phenol,  and  tyrosin.  Systematic  investigations  have  only 
been  made  with  regard  to  the  occurrence  of  indol  and  phenol. 

TEST  FOR  INDOL. — To  a  bouillon  culture,  which  should,  if  possible, 
be  not  under  eight  days  old  and  free  from  sugar,  is  added  half  its  volume 
of  10  per  cent,  sulphuric  acid.  If  in  heating  to  about  80°  C.  a  pink  or 
bluish-pink  coloration  is  immediately  produced  it  indicates  the  presence 
of  both  indol  and  nitrites,  the  above-described  nitroso-indol  reaction 
requiring  the  presence  of  both  of  these  substances  for  its  successful 
operation.  This  is  the  so-called  " cholera-red  reaction,"  but  it  may  be 
applied  to  many  other  spirilla  besides  cholera.  As  a  rule,  however,  the 
addition  of  sulphuric  acid  alone  is  not  sufficient,  and  a  little  nitrite  must 
be  added;  this  may  be  done  later,  the  culture  being  first  warmed  with- 
out nitrite,  when,  if  there  is  no  reaction  or  a  doubtful  one,  1  to  2  c.c. 
of  0.005  per  cent,  solution  of  sodium  nitrite  is  added  until  the  maxi- 
mum reaction  is  obtained.  The  addition  of  strong  solutions  of  nitrite 
colors  the  acid  liquid  brownish-yellow  and  ruins  the  test.  Out  of  sixty 
species  examined  by  Lehmann,  twenty-three  gave  the  indol  reaction. 

Decomposition  of  Fats. — Pure  melted  butter  is  not  a  suitable  culture 
medium  for  bacteria.  The  rancidity  of  butter  is  brought  about  (1)  as 
the  result  of  a  purely  chemical  decomposition  of  the  butter  by  the 
oxygen  of  the  air  under  the  influence  of  sunlight,  and  (2)  through  the 
formation  of  lactic  acid  from  the  milk-sugar  left  in  the  butter.  Fats 
are,  however,  attacked  by  bacteria  when  mixed  with  gelatin  and  used 
as  culture  media,  with  the  consequent  production  of  acid. 

Putrefaction. — By  putrefaction  is  understood  in  common  parlance 
every  kind  of  decomposition  due  to  bacteria  which  results  in  the  pro- 
duction of  malodorous  substances.  Scientifically  considered,  putrefac- 
tion depends  upon  the  decomposition  of  complex  organic  compounds, 


VITAL  PHEXOMEXA  OF  BACTERIA  99 

albuminous  substances,  which  are  frequently  first  peptonized  and  then 
further  decomposed.  Typical  putrefaction  occurs  only  when  oxygen  is 
absent  or  scanty;  the  free  passage  of  air  through  a  culture  of  putrefac- 
tive bacteria — an  event  which  does  not  take  place  in  natural  putrefaction 
—very  much  modifies  the  process :  first,  biologically,  as  the  anaerobic 
bacteria  are  inhibited,  and  then  by  the  action  of  the  oxygen  on  the 
products  or  by-products  of  the  aerobic  and  facultative  anaerobic 
bacteria. 

As  putrefactive  products  we  have  peptone,  ammonia,  and  amines, 
leucin,  tyrosin,  and  other  amido  substances;  oxyfatty  acids,  indol, 
skatol,  phenol,  ptomains,  toxins,  and,  finally,  sulphuretted  hydrogen, 
mercaptans,  carbonic  acid,  hydrogen,  and,  possibly,  marsh-gas. 

Nitrifying  Bacteria. — According  to  recent  observations,  nitrification  is 
produced  by  a  small,  special  group  of  bacteria,  cultivated  with  diffi- 
culty, which  do  not  grow  on  our  usual  culture  media.  From  the  inves- 
tigations of  Winogradsky  it  would  appear  that  there  are  two  common 
micro-organisms  present  in  the  soil,  one  of  which  converts  ammonia 
into  nitrites  and  the  other  converts  nitrites  into  nitrates.  - 

Conversion  of  Nitrous  and  Nitric  Acids  into  Free  Nitrogen. — This  process 
is  performed  by  a  number  of  bacteria.  The  special  nitrate-fermenting 
bacteria,  however,  were  first  accurately  described  by  Barri  and  Stutzer. 
In  their  exhaustive  investigation  they  first  isolated  from  horse-manure 
two  bacteria,  neither  of  which  was  alone  capable  of  producing  nitrogen 
from  nitrates,  but  which  together  in  the  presence  of  oxygen,  but  never 
without  it  entirely,  decomposed  nitrates  energetically.  Later  a  second 
denitrificating  bacillus  was  found,  B.  denitrificans  II,  which  by  itself 
was  able  to  produce  nitrogen  from  nitrates. 

The  practical  importance  of  these  organisms  is  that  by  their  action 
large  quantities  of  nitrates  in  the  soil,  and  especially  in  manure,  may 
become  lost  as  plant  food  by  being  converted  into  nitrogen. 

Nitrogen  Combination. — The  bacillu*  radiocola  of  Beyerinck,  which 
was  isolated  by  him,  has  the  power  of  assimilating  nitrogen  from  the 
air.  This  bacillus  is  found  in  the  small  root  nodules  of  various  legu- 
minous plants  (pease,  clover,  etc.),  and  can  be  obtained  from  these  in 
cultures.  Different  varieties  exist  in  different  kinds  of  legumes,  each 
kind  of  legume  apparently  having  a  special  variety  of  bacteria  adapted 
to  it,  and  not  every  variety  is  capable  of  producing  nodules  in  all  legumes. 
There  are  certain  "neutral"  varieties,  however,  exjtfkg  free  in  the  soil 
and  not  adapted  to  any  special  legume,  and  thd  o  he  able  to 

form  nodules  in  different  legumes. 

By  the  aid  of  these  root  bacteria,  which  e«|pRance  to  the  roots 
and  there  produce  this  nodular  formation,  We  leguminous  plants  are 
enabled  to  assimilate  nitrogen  from  the  atmosphere.  '  This  explains  the 
reason  why  poor,  sandy  soils  become  gradually  fruitful  when  pease, 
lupine,  and  other  varieties  of  legumes  are  grown  upon  them  and  then 
turned  under  with  the  plough.  It  is  not  known  exactly  how  this  assimi- 
lation of  nitrogen  occurs,  but  it  is  assumed  that  the  zoogloea-like  bac- 
teria, called  bacteroids,  constantly  observed  in  the  nodules,  either  alone 


100  PRINCIPLES  OF  BACTERIOLOGY 

or  in  a  special  degree,  possess  the  property  of  assimilating  and  combin- 
ing nitrogen.  It  seems,  moreover,  to  have  been  recently  established 
that,  independently  of  the  assistance  of  the  legumes,  certain  nodule 
bacteria  exist  free  in  the  soil,  which  accumulate  nitrogen  by  absorbing 
it  from  the  air  (Stutzer). 

Formation  of  Acids  from  Carbohydrates. — Free  acids  are  formed  by 
many  bacteria  in  culture  media  containing  sugar;  the  production  of 
acid  in  ordinary  bouillon  takes  place  on  account  of  the  presence  of 
grape-sugar,  which  is  usually  derived  in  small  quantities  from  the  meat.1 
According  to  Theobald  Smith,  all  anaerobic  or  facultative  anaerobic 
bacteria  form  acids  from  sugar;  the  strict  aerobic  species  do  not,  or  so 
very  slowly  that  the  acid  is  concealed  by  the  almost  simultaneous 
production  of  alkali.  The  formation  of  acid  occurs  sometimes  with 
and  sometimes  without  the  production  of  gas.  Excessive  acid  produc- 
tion may  cause  the  death  of  the  bacteria  from  the  increase  in  acidity  of 
the  culture  media. 

If  after  the  sugar  is  consumed  not  enough  acid  has  been  formed  to 
kill  the  bacteria,  the  acid  is  neutralized  gradually  and  in  the  end  the 
reaction  becomes  alkaline. 

Among  the  acids  produced  the  most  important  is  lactic  acid;  also 
traces  of  formic  acid,  acetic  acid,  proprionic  acid,  and  butyric  acid, 
and  not  infrequently  some  ethyl-alcohol  and  aldehyde  or  acetone  are 
formed.  Occasionally  no  lactic  acid  is  present,  and  only  the  other  acids 
are  formed. 

Various  bacteria,  as  yet  incompletely  studied,  possess  the  property 
of  producing  butyric  acid  and  butyl-alcohol  from  carbohydrates. 

Some  bacteria  also  seem  to  have  the  power  of  decomposing  cellulose. 

Formation  of  Gas  from  Carbohydrates  and  Other  Fermentable  Substances 
of  the  Fatty  Series.— The  only  gas  produced  in  visible  quantity  in  sugar- 
free  culture  media  is  nitrogen.  If  sugar  is  vigorously  decomposed  by 
bacteria,  as  long  as  pure  lactic  acid  or  acetic  acid  is  produced  there  may 
be  no  development  of  gas,  as,  for  instance,  with  the  B.  typhosus  on 
grape-sugar;  but  frequently  there  is  much  gas  developed,  especially  in 
the  absence  of  air.  About  one-third  of  the  acid-producing  species  also 
develop  gas  abundantly,  this  consisting  chiefly  of  CO2,  which,  according 
to  Smith,  is  always  mixed  with  H.  Marsh-gas  is  seldom  formed  by 
bacteria,  with  the  exception  of  those  decomposing  cellulose. 

In  order  to  test  the  production  of  gas,  a  culture  medium  composed 
of  glucose-agar,  containing  about  1  per  cent,  grape-sugar,  may  be  used. 
At  the  end  of  eight  to  twelve  hours  in  the  incubator  (or  twenty-four 
hours'  room  temperature)  the  agar  will  be  seen  to  be  full  of  gas-bubbles 
or  broken  up  into  holes  and  fissures. 

For  the  determination  of  the  quantity  and  kind  of  gas  produced  by  a 
given  micro-organism  the  fermentation  tube  recommended  by  Theobald 
Smith  is  the  best.  This  is  a  bent  tube,  constricted  greatly  at  its  lowest 

1  According  to  Theobald  Smith,  75  per  cent,  of  the  beef  ordinarily  bought  in  the  markets  contains 
appreciable  quantities  of  sugar  (up  to  0.3  per  cent.). 


VITAL  PI/l-:.\<>Mi:\A  OF  BACTl.  .,'/.! 


101 


FIG.  64 


portion,   supported   upon   a   glass   base,   as  shown   in   Fig.   64.     The 
tube  is  filled  with  a  culture  media  consisting  of   1   per  cent,  glucose, 
peptone  bouillon  (without  air    bubbles),  and    sterilized   in   the  steam 
sterilizer.     It  is  then  inoculated  with  a  loopful  of  a  cul- 
ture of  the  organism  in  question,  and  observations  taken : 

1.  If  there  is  a  turbidity  produced  in  the  open  bulb 
it  indicates  the  presence  of  an  aerobic  species;  if  this 
clouding  occurs  only  in  the  closed  arm,  while  the  open 
bull)  remains  clear,  it  is  an  anaerobic  species. 

2.  The   quantity  of    gas    produced    daily  should   be 
marked  on  the  upright  arm;  if  the  tube  is  graduated  a 
note  of  it  is  taken  and  the  percentage  calculated  on  the 
fourth  to  the  sixth  day  after  gas  production  has  ceased. 

3.  A  rough    analysis   of   the   gas   produced    may  he 
made   as  follows:  Having  signified   by  a  mark  on   the 
tube   the   quantity  of  gas   produced,  the  open   bulb  is 
completely  filled  with  a  10  per  cent,   solution  of  soda, 
the   mouth    tightly   closed   with    the    thumb,    and    the 
mixture  thoroughly  shaken.     After  a  minute  or  two  all 

the  gas  is  allowed  to  rise  to  the  top  of  the  closed  arm  by  inclining  and 
turning  the  tube,  and  then,  removing  the  thumb,  the  volume  of  gas  left 
after  the  union  of  the  NaOH  with  the  CO2  is  noted.  The  remainder 
is  nitrogen,  hydrogen,  and  marsh-gas.  If  it  is  desired  to  test  for  the 
presence  of  hydrogen,  the  thumb  is  again  placed  over  the  open  end 
and  the  gas  collected  under  it.  As  the  thumb  is  moved  a  lighted  match 
is  brought  in  contact  with  the  gas.  If  hydrogen  is  present  a  slight 
explosion  occurs. 

Formation  of  Acids  from  Alcohol  and  Other  Organic  Acids. — It  has  long 
been  known  that  the  bacterium  aceti  and  other  allied  bacteria  convert 
dilute  solutions  of  ethyl-alcohol,  under  the  influence  of  oxidation,  into 
acetic  acid: 


Fermentation 
tube. 


CH3 
CH2OH 


02 


CH3 
COOH. 


H20. 


The  higher  alcohols — glycerin,  dulcit,  mannite,  etc. — are  also  con- 
verted into  acids — glycerin,  indeed,  as  commonly  as  sugar. 

Finally,  numerous  results  have  been  obtained  from  the  conversion 
of  the  fatty  acids  and  their  salts  into  other  fatty  acids  by  bacteria.  As 
a  rule,  the  lime-salts  of  lactic,  malic,  tartaric,  and  citric  acids  have 
been  employed,  these  being  converted  into  various  acids  by  the  action 
of  bacteria,  such  as  butyric,  proprionic,  valerianic,  and  acetic  acids; 
also  succinic  acid,  ethyl-alcohol,  and,  more  rarely,  formic  acid  have 
been  produced.  Among  the  gases  formed  were  chiefly  CO2  and  H. 

Thus  Pasteur  found  that  anaerobic  bacteria  convert  lactate  of  lime 
into  butyric  acid. 


CHAPTER   VIII. 

THE  EFFECT  OF  VARIOUS  DELETERIOUS  INFLUENCES  UPON 

BACTERIA. 

Influence  of  Electricity  on  Bacteria. — The  majority  of  the  observations 
heretofore  made  on  this  subject  would  seem  to  indicate  that  there  is  no 
direct  action  of  the  galvanic  current  on  bacteria ;  but  the  effect  of  heat 
and  the  electrolytic  influence  on  the  culture  liquid  may  produce  changes 
which  finally  sterilize  it. 

Influence  of  Agitation. — Meltzer  has  shown  that  the  vitality  of  bac- 
teria is  destroyed  by  protracted  and  violent  shaking,  which  causes  a 
disintegration  of  the  cells.  Appel  found  that  moderate  agitation  of  the 
bacteria  caused  no  injury,  even  when  long  continued. 

Influence  of  Pressure. — D'Arsonval  and  Charrin  submitted  a  culture 
of  bacillus  pyocyaneus  to  a  pressure  of  fifty  atmospheres  under  car- 
bonic acid.  At  the  end  of  four  hours  cultures  could  still  be  obtained, 
but  the  bacillus  had  lost  its  power  of  pigment  production.  A  few 
colonies  were  developed  after  six  hours'  exposure  to  this  pressure,  but 
after  twenty-four  hours  no  development  occurred. 

Influence  of  Light. — A  large  number — perhaps  the  majority — of 
bacteria  are  inhibited  in  growth  by  the  action  of  bright  daylight,  all  are 
by  that  of  direct  sunlight,  and  when  the  action  of  the  latter  is  prolonged 
they  lose  their  power  of  developing  when  later  placed  in  the  dark. 

In  order  to  test  the  susceptibility  of  bacteria  to  light,  it  is  best,  accord- 
ing to  H.  Buchner,  to  suspend  a  large  number  of  bacteria  in  nutrient 
gelatin  or  agar  arid  pour  the  media  while  still  fluid  in  Petri  dishes, 
upon  which  has  been  pasted  a  strip  of  black  paper  on  the  side  upon 
which  the  light  is  to  act.  The  action  of  heat  may  be  shut  off  by  allow- 
ing the  ray  of  light  to  first  pass  through  a  layer  of  water  or  alum  of 
several  centimetres'  thickness.  After  the  plates  have  been  exposed  to 
the  light  for  one-half,  one,  one  and  a  half,  two  hours,  etc.,  they  are 
taken  into  a  dark  room  and  allowed  to  stand  at  20°  or  35°  C.  a  sufficient 
length  of  time  to  allow  of  growth,  and  then  examined  to  see  whether 
there  are  colonies  anywhere  except  on  the  spot  covered  by  the  paper; 
when  the  colonies  exposed  to  the  light  have  been  completely  destroyed 
there  is  a  sharply  defined  region  of  the  shape  of  the  paper  strip  crowded 
with  colonies  lying  in  a  clear  sterile  field. 

Dieudonne,  in  experiments  upon  the  bacillus  prodigiosus,  found 
that  direct  sunlight  in  March,  July,  and  August  killed  these  bacilli  in 
one  and  a  half  hours;  in  November  in  two  and  a  half  hours.  Diffuse 
daylight  in  March  and  July  restrained  development  after  three  and  a 
half  hours'  exposure  (in  November  four  and  a  half  hours),  and  com- 


EFFECT  OF  DELETERIOUS  INFLUENCES  UPON  BACTERIA     103 

pletely  destroyed  vitality  in  from  five  to  six  hours.  Electric  arc-light 
inhibited  growth  in  five  hours  and  destroyed  vitality  in  eight  hours. 
Incandescent  light  inhibited  growth  in  from  seven  to  eight  hours  and 
killed  in  eleven  hours.  Similar  results  have  been  obtained  with  B.  coli, 
B.  typhosus,  and  B.  anthracis.  According  to  Koch,  the  tubercle  bacillus 
is  killed  by  the  action  of  direct  sunlight  in  a  time  varying  from  a  few 
minutes  to  several  hours,  depending  upon  the  thickness  of  the  layer 
exposed  and  the  season  of  the  year.  Diffuse  daylight  also  had  the  same 
effect,  although  a  considerably  longer  time  of  exposure  was  required— 
when  placed  close  to  a  window,  from  five  to  seven  days. 

Only  the  ultraviolet,  violet,  and  blue  rays  of  the  spectrum  seem  to 
possess  bactericidal  action ;  green  light  has  very  much  less  so ;  red  and 
yellow  light  not  at  all.  The  action  of  light  is  apparently  assisted  by  the 
admission  of  air;  anaerobic  species,  like  the  tetanus  bacillus,  and  facul- 
tative anaerobic  species,  such  as  the  colon  bacillus,  are  able  to  withstand 
quite  well  the  action  of  sunlight  in  the  absence  of  oxygen,  the  B.  coli 
intense  direct  sunlight  for  four  hours. 

According  to  Richardson  and  Dieudonne,  the  mechanism  of  the 
action  of  light  may  be  at  least  partially  explained  by  the  fact  that  in 
agar  plates  exposed  to  light  for  a  short  time  (even  after  ten  minutes' 
exposure  to  direct  sunlight)  hydrogen  peroxide  (H2O2)  is  formed.  This 
is  demonstrated  by  exposing  an  agar  plate  half  covered  with  black 
paper,  upon  which  a  weak  solution  of  iodide  of  starch  is  poured,  and 
over  this  again  a  dilute  solution  of  sulphate  of  iron;  the  side  exposed 
to  the  light  turns  blue-black.  In  gases  containing  no  oxygen,  hydrogen 
peroxide  is  not  produced,  and  the  light  has  no  injurious  effect.  Access 
of  oxygen  also  explains  the  effect  which  light  produces  on  culture  media 
which  have  been  exposed  to  the  action  of  sunlight,  as  standing  in  the 
sun  for  a  time,  when  afterward  used  for  inoculation.  The  bacteria 
subsequently  introduced  into  such  media  grow  badly — far  worse  than 
in  fresh  culture  media  which  are  kept  in  the  shade. 

Influence  of  Radium. — Radioactive  fluids  have  a  slight  inhibiting 
effect  on  bacterial  growth,  but  nothing  decided  enough  to  be  used  for 
therapeutic  purposes. 

Influence  of  X-rays. — These  rays  have  a  slight  inhibiting  effect  on 
bacteria  when  they  are  directly  exposed  to  them. 

Influence  of  One  Species  upon  the  Growth  of  Another. — While  it  is  the 
custom  of  bacteriologists  to  have  pure  cultures  to  work  with,  we  should 
never  forget  that  in  nature  bacteria  often  occur  in  mixed  cultures.  If 
we  examine  water,  milk,  or  the  contents  of  the  intestines  of  either  sick 
or  healthy  persons  we  shall  always  find  several  species  of  bacteria  occur- 
ring together.  This  admixture  may,  perhaps,  seem  to  us  at  first  merely 
accidental,  but  on  further  investigation  it  will  appear  also  that  in  the 
department  of  bacteriology  there  exist  synergists  and  antagonists,  or 
at  least  bacteria  which  assist  or  oppose  one  another  mutually  or  one- 
sidedly.  Nencki  speaks  of  symbiosis  and  enantobiosis. 

The  existence  of  antagonisms  can  be  demonstrated  experimentally 
by  inoculating  gelatin  streak  cultures  of  various  bacteria.  It  is  found 


104  PRINCIPLES  OF  BACTERIOLOGY 

that  many  species  will  not  grow  at  all  or  only  sparingly  when  in  close 
proximity  to  some  other  species.  This  antagonism,  however,  is  often 
only  one-sided  in  character;  for  instance,  the  bacillus  fluorescens  putidus 
grows  well  when  inoculated  between  streaks  of  staphylococcus,  but 
the  latter  micrococcus  will  not  grow  at  all  when  inoculated  between 
cultures  of  the  bacillus  putidus,  the  growth  of  the  staphylococcus  remain- 
ing scanty  when  the  two  species  are  inoculated  simultaneously.  Again, 
when  gelatin  or  agar  plates  are  made  from  two  different  species  of 
bacteria  it  may  be  observed  that  only  one  of  the  two  grows.  A  third 
method  of  making  this  experiment  is  to  simultaneously  inoculate  the 
same  liquid  medium  with  two  species,  and  then  examine  them  later, 
both  microscopically  and  by  making  plate  cultures;  not  infrequently 
the  one  species  may  take  precedence  of  the  other,  which  it  finally 
overcomes  entirely.  The  practical  application  of  this  is  to  make 
sufficient  dilutions  of  material  when  plating  for  the  estimation  of  the 
number  of  bacteria  or  the  isolation  of  pure  cultures. 

Finally,  bacteria  may  oppose  one  another  as  antagonists  in  the  animal 
body.  As  Emmerich  has  shown,  animals  infected  with  anthrax  may 
often  be  cured  by  a  secondary  infection  with  the  streptococcus. 

The  symbiotic  or  co-operative  action  of  bacteria  is  of  still  greater 
importance,  of  which  the  following  examples  may  be  given: 

1.  Some  bacteria  thrive  better  in  association  with  other  species  than 
alone.     Brueger  has  recently  shown  that  pneumococci  when  grown 
together  with  a  bacillus  obtained  from  the  throat,  produce  very  large, 
succulent  colonies.      Certain   anaerobic   species    grow  even   with   the 
admission  of  air  if  only  other  aerobic  species  are  present  (tetanus). 

2.  Certain  chemical  effects,  as,  for  instance,  the  decomposition  of 
nitrates  to  gaseous  nitrogen,  cannot  be  produced  by  many  bacteria 
alone,  but  only  when  two  are  associated. 

3.  Attenuated  varieties  of  bacteria  may  regain  virulence  when  grown 
in  contact  with  other  bacteria. 

Duration  of  Life  in  Pure  Water. — When  bacteria  which  require  much 
organic  food  for  their  development,  and  these  include  most  of  the  path- 
ogenic species,  are  placed  in  distilled  water  they  soon  die — that  is, 
within  a  few  days;  even  in  sterilized  well  water  or  surface  water  their  life 
duration  does  not  usually  exceed  eight  to  fourteen  days,  and  they  rarely 
multiply.  Instances,  however,  of  much  more  extended  life  under  certain 
conditions  are  recorded. 

Effect  of  Drying. — Want  of  water  affects  bacteria  in  different  ways. 
Upon  dried  culture  media  development  soon  ceases;  but  in  media  dried 
gradually  at  the  room  temperature  (nutrient  agar,  gelatin,  potato)  they 
live  often  for  a  long  time,  even  when  there  are  no  spores  to  account  for 
it.  A  shrunken  residue  of  such  cultures  in  bouillon  has  often  been 
found,  after  a  year  or  more,  to  yield  living  bacteria.  The  question  as 
to  how  long  the  non-spore  bearing  forms  are  capable  of  retaining  their 
vitality  when  dried  on  a  cover-glass  or  silk  threads  has  been  variously 
answered.  We  know  now  that  there  are  many  factors  which  influence 
the  retention  of  vitality.  The  following  table  of  the  results  obtained 


/•:/-7-7-;rr  OF  DELETERIOUS  i\  ri.  i  'i-:\<  'ES  i  'r<  >\  n.  i  (  ' 


A    105 


by  Siivna  and  Alessi  gives  some  idea  of  the  extent  and  effect  of  such 
influences.  In  the  experiments  silk  threads  were  saturated  with  bouillon 
cultures  or  aqueous  suspensions  of  the  bacteria,  and  some  then  enclosed 
in  tubes  containing  sulphuric  acid  or  calcium  chloride,  wrhile  others 
were  left  exposed  to  various  outside  influences: 


With  sulphuric    With  calcium     In  incubator     In  dry  room  in  i     In  moist 
Desiccation.  acid,  killed  at  chloride,  killed    at  37°,  killed      shade,  killed     room,  killed 

end  of  at  end  of  at  end  of  at  end  of  at  end  of 


Cholera  spirilla   .    . 

Iday 

Iday 

Iday 

Iday 

12  days 

B.  of  fowl  cholera   . 

2  days 

1    " 

1    " 

5  days              59    " 

B.  typhosus  .... 

41     "                    1    " 

18  days 

64    •• 

68    " 

B.  mallei     .... 

35     ••                   44  days 

31    " 

Diplococ.  pneumonise 

114      "                    31    "                   131    " 

164    "                 192    " 

The  results  of  all  investigators,  however,  would  seem  to  indicate 
that  the  greatest  possible  care  must  be  exercised  in  desiccation  experi- 
ments to  come  to  any  positive  conclusions ;  but  recently  most  astonishing 
results  have  been  obtained  with  regard  to  many  species  usually  sup- 
posed to  be  particularly  sensitive  to  desiccation,  showing  that  under 
certain  conditions  they  may  retain  their  vitality  in  a  dry  state  for  a 
very  long  time.  Thus,  Koch  found  that  cholera  spirilla  lived  only  a 
few  hours  when  dry;  Kitasato  determined  their  life  duration  at  four- 
teen days  at  most;  while  various  French  observers  have  found  that 
they  may,  under  favorable  conditions,  live  150  to  200  days.  The  vary- 
ing results  sometimes  reported  by  different  observers  in  such  experi- 
ments may  be  explained  by  the  fact  that  the  conditions  under  which 
they  were  made  were  different,  depending  upon  the  desiccator  used, 
the  medium  upon  which  the  cultures  were  grown,  and  the  use  of  silk 
threads  or  cover-glasses.  In  all  these  experiments,  of  course,  it  should 
be  previously  determined  that  in  spore-bearing  species  there  are  no 
spores  present.  Even  when  a  dried  culture  lives  for  a  long  time  the 
majority  of  the  organisms  die  in  a  few  hours  after  drying.  We  have 
found  1,500,000  colon  bacilli  to  be  reduced  to  100^000  after  three 
hours'  drying.  \Vhen  protected  by  a  covering  of  mucus,  as  in  expec- 
toration, they  live  much  longer  than  when  unprotected. 

Behavior  toward  Oxygen  and  Other  Gases. — As  already  noted  under 
the  nutritious  substances  required  by  bacteria,  it  is  customary  to  divide 
bacteria  into  three  classes,  according  to  their  behavior  toward  oxygen. 

1.  AEROBIC  BACTERIA. — Growth  only  in  the  presence  of  oxygen: 
the  slightest  restriction  of  air  inhibits  development.     Spore  formation 
especially  requires  the  free  admission  of  air. 

2.  ANAEROBIC  BACTERIA. — Growth  and  spore  formation  only  in  the 
total  exclusion  of  oxygen.    Among  this  class  of  bacteria  are  the  bacillus 
of  malignant  oedema,  the  tetanus  bacillus,  the  bacillus  of  symptomatic 
anthrax,  and  many  soil  bacteria.     Exposed  to  the  action  of  oxygen, 
the  vegetative  forms  of  these  bacteria  are  readily  destroyed ;  the  spores, 


106  PRINCIPLES  OF  BACTERIOLOGY 

on  the  contrary,  are  very  resistant.  Anaerobic  bacteria  being  deprived 
of  oxygen — the  chief  source  of  energy  supplied  to  the  aerobic  species, 
by  which  they  oxidize  the  nutritive  substances  in  the  culture  media — 
they  are  dependent  for  their  nutrition  upon  decomposable  substances, 
such  as  grape-sugar,  which  on  separating  into  two  smaller  molecules, 
alcohol  and  carbonic  acid,  give  out  energy  or  heat.  Anaerobic  bacteria, 
therefore,  require  for  their  cultivation,  as  a  rule,  media  containing 
glucose  or  some  equivalent. 

3.  FACULTATIVE  AEROBIC  AND  FACULTATIVE  ANAEROBIC  BACTERIA. 
—The  greater  number  of  aerobic  bacteria,  including  most  of  the  path- 
ogenic species,  are  capable  of  withstanding,  without  being  seriously 
affected,  some  restriction  in  the  amount  of  oxygen  admitted,  and  many, 
indeed,  grow  equally  luxuriantly  in  the  partial  exclusion  of  oxygen. 
Life  in  the  animal  body,  for  example,  as  in  the  intestines,  necessitates 
existence  with  diminished  supply  of  oxygen.  Pigment  formation  almost 
always  ceases  with  the  exclusion  of  oxygen,  but  poisonous  products  of 
decomposition  may  be  more  abundantly  produced. 

It  is  important  to  note  that,  according  to  recent  investigations,  it 
has  been  shown  that  the  aerobic  development  of  the  anaerobes  may 
be  facilitated  by  the  presence  of  living  or  dead  aerobes. 

It  has  also  been  observed  not  infrequently  that  certain  species  which 
on  their  isolation  at  first  showed  more  or  less  anaerobic  development 
— that  is,  a  preference  to  grow  in  the  depth  of  an  agar  stick  culture, 
for  instance — after  a  while  seem  to  become  strict  aerobes,  growing  only 
on  the  surface  of  the  medium.  This  observation  proves  that  the  simple 
fact  of  an  organism  showing  aerobic  instead  of  anaerobic  growth  is  not 
sufficient  for  its  separation  into  a  distinct  species. 

While  all  facultative  as  well  as  strict  anaerobes  grow  well  in  nitrogen 
and  hydrogen,  they  behave  very  differently  toward  carbonic  acid  gas. 
A  large  number  of  these  species  do  not  grow  at  all,  being  completely 
inhibited  in  their  development  until  oxygen  is  again  admitted — for 
example,  B.  anthracis  and  B.  subtilis  and  other  allied  species.  It  has 
been  found  in  some  species,  as  glanders  and  cholera,  that  the  majority 
of  the  organisms  are  quickly  killed  by  CO2,  while  a  few  offer  a  great 
resistance,  rendering  impossible  complete  sterilization  by  means  of 
this  gas.  Another  group,  again — viz.,  streptococcus  and  staphylo- 
coccus — exhibits  a  scanty  growth;  while  a  third  group,  like  the  B. 
typhosus  and  B.  prodigiosus,  is  not  at  all  affected,  growing  equally  as 
well  in  the  presence  of  oxygen,  and  the  liquefaction,  even  of  gelatin, 
not  being  interfered  with;  only,  on  account  of  the  lack  of  oxygen,  there 
is  no  pigment  formation.  Finally,  a  mixture  of  one-fourth  air  to  three- 
fourths  carbonic  acid  gas  seems  to  have  no  injurious  effect  on  bacteria 
which  cannot  grow  in  an  atmosphere  of  pure  CO2. 

Sulphuretted  hydrogen  in  large  quantity  is  a  strong  bacterial  poison, 
and  even  in  small  amounts  kills  some  bacteria. 


CHAPTER    IX. 

THE  DESTRUCTION  OF  BACTERIA  BY  CHEMICALS— PRACTICAL 
USE  OF  DISINFECTANTS. 

MANY  chemical  substances,  when  brought  in  contact  with  bacteria, 
unite  with  their  cell  substance.  New  compounds  are  thus  formed,  and 
the  life  of  the  bacteria  and  the  disinfecting  properties  of  the  substances 
art'  usually  destroyed;  while  in  the  vegetative  stage  bacteria  are  much 
more  easily  killed  than  when  in  the  spore  form,  and  their  life  processes 
are  inhibited  by  substances  less  deleterious  than  those  required  to 
destroy  them. 

Bacteria,  both  in  the  vegetative  and  in  the  spore  form,  differ  among 
themselves  considerably  in  their  resistance  to  the  poisonous  effects  of 
chemicals.  The  reason  for  this  is  not  as  yet  clear,  but  is  apparently 
connected  with  the  structure  and  chemical  nature  of  their  cell  substance. 

Chemicals  are  more  poisonous  at  fairly  high  than  at  a  low  tempera- 
ture, and  act  more  quickly  upon  bacteria  when  they  are  suspended  in 
fluids  singly  than  when  in  clumps,  and  in  pure  water  rather  than  in 
solutions  containing  organic  matter.  The  increased  energy  of  disin- 
fectants at  higher  temperatures  indicates  in  itself  a  probability  that  a 
true  chemical  reaction  takes  place.  In  estimating  the  extent  of  the 
destructive  action  of  chemicals  the  following  degrees  are  usually  dis- 
tinguished: 

1.  The  growth  is  not  permanently  interfered  with,  but  the  pathogenic 
and  zvmogenic  functions  of  the  organism  are  diminished — attenuation. 

2.  The  organisms  are  not  able  to  multiply,  but  they  are  not  destroyed 
by  antiseptic  action. 

3.  The  vegetative  development  of  the  organisms  is  destroyed,  but 
not  the  spores — incomplete  sterilization. 

4.  Vegetative  and  spore  formation  are  destroyed.    This  is  complete 
sterilization  or  disinfection.1 

The  methods  employed  for  the  determination  of  the  germicidal 
action  of  chemical  agents  on  bacteria  are,  briefly,  as  follows : 

If  it  is  desired  to  determine  what  is  the  minimum  concentration  of 
the  chemical  substance  required  to  produce  complete  inhibition  of 
growth  we  proceed  thus:  A  10  per  cent,  solution  of  the  disinfectant  is 
prepared  and  1  c.c.,  0.5  c.c.,  0.3  c.c.,  0.1  c.c.,  etc.,  of  this  is  added  to 
10  c.c.  of  liquefied  gelatin,  agar,  or  bouillon,  or,  more  accurately,  10  c.c. 

1  Disinfection  strictly  defined  is  the  destruction  of  all  organisms  and  their  products  which  are 
capable  of  producing  disease.  Sterilization  is  the  destruction  of  all  saprophytic  as  well  as  parasitic 
bacteria.  Practically,  however,  the  two  terms  are  used  interchangeably  as  meaning  the  destruction 
of  all  living  bacteria. 


108  PRINCIPLES  OF  BACTERIOLOGY 

minus  the  amount  of  solution  added,  in  so  many  tubes.  The  tubes  then 
contain  1  per  cent.,  0.5  per  cent.,  0.3  per  cent.,  and  0.1  per  cent,  of  the 
disinfectant.  The  fluid  media  in  the  tubes  are  then  inoculated  with  a 
platinum  loopful  of  the  test  bacteria.  The  melted  agar  and  gelatin 
may  be  simply  shaken  and  allowed  to  remain  in  the  tubes,  and  watched 
as  to  whether  any  growth  takes  place,  or  the  contents  of  the  tubes  are 
poured  out  into  Petri  dishes,  where  the  development  or  lack  of  devel- 1 
opment  of  colonies  and  the  number  can  be  observed.  The  same  test 
can  be  made  with  material  containing 'only  spores. 

If  it  is  desired  to  determine  the  degree  of  concentration  required 
for  the  destruction  of  vegetative  development,  the  organism  to  be  used 
is  cultivated  in  bouillon,  and  to  each  of  a  series  of  tubes  holding  in 
watery  solution  different  percentages  of  the  disinfectant  a  few  drops 
of  the  culture  from  which  all  lumps  have  been  filtered  are  added.  At 
intervals  of  one,  five,  ten,  fifteen,  and  thirty  minutes,  one  hour,  and  so 
on  a  small  platinum  loopful  of  the  mixture  is  taken  from  each  tube 
and  inoculated  into  10  c.c.  of  fluid  agar  or  gelatin,  from  which  plate  cul- 
tures are  made.  The  results  obtained  are  signified  as  follows:  x  per 
cent,  of  the  disinfectant  in  watery  solution  and  at  x  temperature  kills 
the  organism  in  twenty  minutes,  y  per  cent,  kills  in  one  minute,  and 
so  on.  If  there  be  any  doubt  whether  the  trace  of  the  disinfectant  car- 
ried over  with  the  platinum  loops  may  have  rendered  the  gelatin  unsuit- 
able for  growth,  thus  falsifying  results,  control  cultures  should  be  made 
with  vigorous  bacteria  in  gelatin  to  which  a  similar  trace  of  the  disin- 
fectant has  been  added.  If  the  strength  of  the  disinfectant  is  to  be 
discovered  in  different  substances  it  must  be  tested  in  these  substances 
and  not  in  water. 

The  disinfectant  to  be  examined  should  always  be  dissolved  in  an 
inert  fluid,  such  as  water;  if  on  account  of  its  being  difficultly  soluble  in 
water,  it  is  necessary  to  use  alcohol  for  its  solution,  control  experiments 
may  be  required  to  determine  the  action  of  the  alcohol  on  the  organism „ 
Sometimes,  as  in  the  case  of  corrosive  sublimate,  the  chemical  unites 
with  the  cell  substance  to  form  an  unstable  compound,  which  inhibits 
the  growth  of  the  organism  only  while  the  union  exists.  If  this  com- 
pound is  not  broken  up  in  the  media,  it  will  probably  not  be  in  the 
body.  In  some  tests  it  is  of  interest  to  break  up  this  union  and  note 
then  whether  the  organism  is  alive  or  dead. 

In  the  above  determinations  the  absolute  strength  of  the  disinfectant 
required  is  considerably  less  when  culture  media  rich  in  albumin  are 
employed  than  when  the  opposite  is  the  case.  Cholera  spirilla  grown 
in  bouillon  containing  no  peptone  or  only  0.5  per  cent,  of  peptone  are 
destroyed  in  half  an  hour  by  0.1  per  cent,  of  hydrochloric  acid;  grown 
in  2  per  cent,  peptone-bouillon  their  vitality  is  destroyed  in  the  same 
time  on  the  addition  of  0.4  per  cent.  HC1.  In  any  case  the  organisms 
to  be  tested  should  all  be  treated  in  exactly  the  same  way  and  the  results 
accompanied  by  a  statement  of  the  conditions  under  which  the  tests 
were  made. 


THE  DESTRUCTION  OF  BACTERIA  BY  CHEMICALS 


109 


The  following  table  gives  the  results  and  methods  used  in  an  actual 
experiment  to  test  the  effect  of  blood  serum  upon  the  disinfecting  action 
of  bichloride  of  mercury  and  carbolic  acid  upon  bacteria: 

TEST  FOR  THE  DIFFERENCE  OF  EFFECT  OF  BICHLORIDE  OF  MERCURY  AND 
CARBOLIC  ACID  SOLUTIONS  ON  ANTHRAX  AND  TYPHOID  BACILLI  IN  SERUM 
AND  BOUILLON. 


Time    .    .    . 

1' 

3' 

y 

IV 

20' 

30' 

45' 

Ihr. 

l^hrs.    2hrs. 

A.  Serum  .  .  .  2.5  c.c.) 
HgCl-  sol.  1  :  1000  2.5  c  c.  V 
Anthrax  threads  .  .  .j 

+ 

+ 

+ 

+ 

-f 

+ 

- 

(  Solution  equals 
—            —       4         1  :  -2000 
(     bichloride. 

B.  Bouillon  .  .  2.5  c.c.) 
HgClo  sol.  1  :  1000  2.5  c.c.  V 
Anthrax  threads  .  .  .) 

C.  Serum  .  .  2.5  c.c.) 
pRrhnlin  <?nl  T  ty  2  fi  <»  n_  > 

- 

— 

- 

- 

- 

—         Same. 

(  Solution  equals 
•s  %y  %  carbolic 

Typhoid  threads  .  .  .) 

1         acid. 

D.  Bouillon  .  .  2.5  c.c.) 
Carbolic  sol.  5  %  2.5  c.c.  > 
Typhoid  threads  .  .  .) 

+ 

- 

+ 

+ 

+ 

+ 

- 

- 

—          Same. 

—  Indicates  total  destruction  of  bacteria  with  no  growth  in  media. 
+  Indicates  lack  of  destruction  of  bacteria  with  growth  in  media. 

Pieces  of  sterile  thread  (one  inch)  were  placed  in  bouillon  cultures 
of  anthrax  and  typhoid  bacilli  for  ten  minutes,  then  removed  to  Petri 
dishes,  and  dried  in  the  incubator  for  twenty-four  hours.  These  were 
then  placed  in  serum  and  bouillon  respectively  (2.5  c.c.).  From  each 
a  control  was  taken.  Then  2.5  c.c.  HgCl2  (1 :1000)  and  carbolic  solution 
(5  per  cent.)  was  added  to  either,  as  shown  in  A,  B,  C,  and  D.  From 
each  one  thread  was  taken  at  varying  periods  of  time  and  planted  in 
bouillon  tubes.  The  threads  from  A  and  B  (HgCl2  solution)  were 
washed  in  sterile  water,  then  in  a  solution  of  ammonium  sulphide  (25 
per  cent.)  to  remove  the  HgCl2,  then  in  sterile  water  again,  then  in  the 
bouillon.  The  threads  from  the  carbolic  solution  were  washed  in 
sterile  water  before  planting. 

Observations:  The  serum  seems  to  have  an  inhibitory  action  with 
the  bichloride  solution,  allowing  a  growth  up  to  forty-five  minutes,  while 
with  the  bouillon  the  action  is  much  quicker,  preventing  a  growth  after 
an  exposure  of  one  minute  or  over.  With  the  carbolic  acid  solution  the 
serum  made  less  difference  in  the  results. 

Many  substances  which  are  strong  disinfectants  become  altered 
under  the  conditions  in  which  they  are  used,  so  that  they  lose  a  portion 
or  all  of  their  germicidal  properties;  thus,  quicklime  and  milk  of  lime 
act  by  means  of  their  alkali  and  are  disinfecting  agents  only  so  long  as 
sufficient  calcium  hydroxide  is  present.  If  this  is  changed  by  the  carbon 
dioxide  of  the  air  into  carbonate  of  lime  it  becomes  harmless.  Bichloride 
of  mercury  and  many  other  chemicals  form  compounds  with  many 
organic  and  inorganic  substances,  which,  though  still  germicidal,  are 
much  less  so  than  the  original  substances. 


HO  PRINCIPLES  OF  BACTERIOLOGY 

The  Disinfecting  Properties  of  the  More  Commonly  Used 

Disinfectants. 

Bichloride  of  Mercury. — This  substance,  when  present  in  1  part  in 
1,000,000  nutrient  gelatin  or  bouillon,  prevents  the  development  of 
parasitic  bacteria.  In  water  1  part  in  500,000  will  kill  many  varieties 
in  a  few  minutes,  but  in  bouillon  twenty-four  hours  may  be  needed. 
With  organic  substances  its  power  is  lessened,  so  that  1  part  to  1000 
may  be  required.  Most  spores  are  killed  in  1 : 1000  watery  solution 
within  one  hour.  Corrosive  sublimate,  as  already  noted,  is  less  effective 
as  a  germicide  in  alkaline  fluids  containing  much  albuminous  substance 
than  in  watery  solution.  In  such  fluids,  besides  loss  in  other  ways, 
precipitates  of  albuminate  of  mercury  are  formed  which  are  at  first 
insoluble,  so  that  a  part  of  the  mercuric  salt  does  not  really  exert  any 
action.  In  alkaline  solutions,  such  as  blood,  blood  serum,  pus,  tissue 
fluids,  etc.,  the  soluble  compounds  of  mercury  are  converted  into  oxides 
or  hydroxides.  The  soluble  compounds  can,  of  course,  remain  in  solu- 
tion only  when  there  are  present  sufficient  quantities  of  certain  bodies 
which  render  solution  possible.  Bodies  of  this  sort  are  especially 
the  alkaline  chlorides  and  iodides,  and,  above  all,  sodium  chloride  and 
ammonium  chloride.  A  very  simple  way  of  preventing  precipitation 
of  the  mercury,  then,  is  to  add  a  suitable  quantity  of  common  salt  to 
the  corrosive  sublimate.  Those  compounds  of  mercury  which,  like 
the  cyanides,  are  not  precipitated  with  alkalies,  because  they  at  once 
form  double  salts,  require  no  addition  of  salt. 

For  ordinary  use,  where  corrosive  sublimate  is  employed,  solutions 
of  1 : 500  and  1 : 1000  will  suffice,  when  brought  in  contact  with 
bacteria  in  that  strength,  to  kill  the  vegetative  forms  within  fifteen 
minutes,  the  stronger  solution  to  be  used  when  much  organic  matter 
is  present. 

Biniodide  of  Mercury. — This  salt  is  very  similar  in  its  effects  to  the 
bichloride.  It  is  somewhat  more  powerful. 

Nitrate  of  Silver. — Nitrate  of  silver  in  solution  has  about  one-fourth 
the  value  of  the  bichloride  of  mercury  as  a  disinfectant,  but  nearly  the 
same  value  in  inhibiting  growth. 

Sulphate  of  Copper. — This  salt  has  about  50  per  cent,  of  the  value  of  j 
mercuric  chloride.  It  has  a  quite  remarkable  affinity  for  many  forms  of 
algae,  so  that  when  in  water  1 : 1,000,000  it  destroys  many  forms;  1 : 400,000 
destroys  typhoid  bacilli  in  twenty-four  hours  when  the  water  has  no 
excessive  amount  of  organic  material.  It  is  not  known  to  be  poisonous 
in  this  strength,  so  that  it  can  be  temporarily  added  to  water  supplies. 

Sulphate  of  Iron. — This  is  a  much  less  powerful  disinfectant  than 
sulphate  of  copper. 

Sodium  Compounds.— A  30  per  cent,  solution  of  NaOH  kills  anthrax 
spores  in  about  ten  minutes,  and  in  4  per  cent,  in  about  forty-five  minutes. 
Sodium  carbonate  kills  spores  with  difficulty  even  in  concentrated  solu- 
tion, but  at  85°  C.  it  kills  spores  in  from  eight  to  ten  minutes.  A  5  per 
cent,  solution  kills  in  a  short  time  the  vegetative  forms  of  bacteria. 


THE  DESTRUCTION  OF  BACTERIA  BY  CHEMICALS  111 

Even  ordinary  soapsuds  have  a  slight  bactericidal  as  well  as  a  marked 
cleansing  effect.  The  bicarbonate  has  almost  no  destructive  effect  on 
bacteria. 

Calcium  Compounds. — Calcium  hydroxide  Ca(OH)2  is  a  powerful 
disinfectant;  the  carbonate,  on  the  other  hand,  is  almost  of  no  effect. 
A  1  per  cent,  watery  solution  of  the  hydroxide  kills  bacteria  which  are 
not  in  the  spore  form  within  a  few  hours.  A  3  per  cent,  solution  kills 
typhoid  bacilli  in  one  hour.  A  20  per  cent,  solution  added  to  equal 
parts  of  feces  or  other  filth  and  mixed  with  them  will  completely  sterilize 
them  within  one  hour. 

Effect  of  Acids. — An  amount  of  acid  which  equals  40  c.c.  of  normal 
hydrochloric  acid  per  litre  is  sufficient  to  prevent  the  growth  of  all 
varieties  of  bacteria  and  to  kill  many.  Twice  this  amount  destroys  most 
bacteria  within  a  short  time.  The  variety  of  acid  makes  little  difference. 
Bulk  for  bulk,  the  mineral  acids  are  more  germicidal  than  the  vegetable 
acids,  but  that  is  because  their  molecular  weight  is  so  much  less.  A 
1 : 500  solution  of  sulphuric  acid  kills  typhoid  bacilli  within  one  hour. 
Hydrochloric  acid  is  about  one-third  weaker,  and  acetic  acid  some- 
what weaker  still.  Citric,  tartaric,  malic,  formic,  and  salicylic  acids 
are  similar  to  acetic  acid.  Boric  acid  destroys  the  less  resistant  bac- 
teria in  2  per  cent,  solution  and  inhibits  the  others. 

Gaseous  Disinfectants. 

The  germicidal  action  of  gases  is  much  more  active  in  the  presence 
of  moisture  than  in  a  dry  condition. 

Numerous  experiments  have  been  made  with  sulphur  dioxide  gas 
(SO,'),  owing  to  the  fact  that  it  has  been  so  extensively  used  for  the 
disinfection  of  hospitals,  ships,  apartments,  clothing,  etc.  This  gas 
is  a  much  more  active  germicide  in  a  moist  than  in  a  dry  condition; 
due,  no  doubt,  to  the  formation  of  the  more  active  disinfecting  agent 
— sulphurous  acid  (H2SO3).  In  a  pure  state  anhydrous  sulphur  dioxide 
does  not  destroy  spores,  and  is  not  certain  to  destroy  bacteria  not  in 
spore  form.  Sternberg  has  shown  that  the  spores  of  the  bacillus  anthracis 
and  bacillus  subtilis  are  not  killed  by  contact  for  some  time  with  liquid 
SO0  ( liquefied  by  pressure).  Koch  found  that  various  species  of  spore- 
bearing  bacilli  exposed  for  ninety-six  hours  in  a  disinfecting  chamber 
to  the  action  of  SO2,  in  the  proportion  of  from  4  to  6  per  cent,  by  volume, 
were  not  destroyed.  In  the  absence  of  spores,  however,  the  anthrax 
bacillus  in  a  moist  condition,  attached  to  silk  threads,  was  found  by 
Sternberg  to  be  destroyed  in  thirty  minutes  in  an  atmosphere  containing 
1  volume  per  cent.  As  the  result  of  a  large  number  of  experiments 
with  SO2  as  a  disinfectant  it  has  been  determined  that  an  "exposure 
for  eight  hours  to  an  atmosphere  containing  at  least  4  volumes  per 
cent,  of  this  gas  in  the  presence  of  moisture"  will  destroy  most,  if  not 
all,  of  the  pathogenic  bacteria  in  the  absence  of  spores.  Four  pounds 
of  sulphur  burned  for  each  1000  cubic  feet  will  give  an  excess  of 
gas. 


112  PRINCIPLES  OF  BACTERIOLOGY 

Peroxide  of  hydrogen  (H2O2)  is  an  energetic  disinfectant,  and  in  2 
per  cent,  solution  (about  40  per  cent,  of  the  ordinary  commercial 
article)  will  kill  the  spores  of  anthrax  in  from  two  to  three  hours.  A 
20  per  cent,  solution  of  a  good  commercial  hydrogen  peroxide  solution 
will  quickly  destroy  the  pyogenic  cocci  and  bther  spore-free  bacteria. 
It  combines  with  organic  matter,  becoming  inert.  It  is  prompt  in  its 
action  and  not  poisonous,  but  apt  to  deteriorate  if  not  properly  kept. 

Chlorine. — Chlorine  is  a  powerful  gaseous  germicide,  owing  its  activity 
to  its  affinity  for  hydrogen  and  the  consequent  release  of  nascent  oxygen 
when  it  comes  in  contact  with  micro-organisms  in  a  moist  condition. 
It  is,  therefore,  a  much  more  active  germicide  in  the  presence  of  moisture 
than  in  a  dry  condition.  Thus,  Fischer  and  Proskauer  found  that 
dried  anthrax  spores  exposed  for  an  hour  in  an  atmosphere  containing 
44.7  per  cent,  of  dry  chlorine  were  not  destroyed;  but  if  the  spores  were 
previously  moistened  and  were  exposed  in  a  moist  atmosphere  for  the 
same  time,  4  per  cent,  was  effective,  and  when  the  time  was  extended 
to  three  hours  1  per  cent,  destroyed  their  vitality.  The  anthrax  bacillus, 
in  the  absence  of  spores,  was  killed  by  exposure  in  a  rnoist  atmosphere 
containing  1  part  to  2500  for  twent}^-four  hours. 

In  watery  solutions  0.2  per  cent,  kills  spores  within  five  minutes  and] 
the  vegetative  forms  almost  immediately. 

Chloride  of  Lime. — The  efficacy  of  chloride  of  lime  depends  on  the 
chlorine  it  contains  in  the  form  of  hypochlorites.  A  solution  in  water 
of  0.5  to  1  per  cent,  of  chloride  of  lime  will  kill  most  bacteria  in  one  to 
five  minutes.  A  5  per  cent,  solution  usually  destroys  spores  within  one 
hour. 

Bromine  and  iodine  are  of  about  the  same  value  as  chlorine  foij 
gaseous  disinfectants,  in  the  moist  condition;  but,  like  chlorine,  they 
are  not  applicable  for  general  use  in  house  disinfection,  owing  to  their 
poisonous  and  destructive  properties;  they  have  a  use  in  sewers  and 
similar  places. 

Trichloride  of  iodine  in  0.5  per  cent,  solution  destroys  the  vegetative; 
forms  of  bacteria  in  five  minutes. 

Organic  Disinfectants. 

Alcohol  in  10  per  cent,  solution  inhibits  the  growth  of  bacteria; 
absolute  alcohol  kills  bacteria  in  the  vegetative  fornT~>n  from  several 
to  twenty-four  hours. 

Formaldehyde. — Formaldehyde,  or  formic  aldehyde,  was  isolated 
by  von  Hoffmann  in  1867,  who  obtained  it  by  passing  the  vapors  of 
methyl-alcohol  mixed  with  air  over  finely  divided  platinum  heated  to 
redness.  The  methyl-alcohol  is  oxidized  and  produces  formaldehyde 
as  follows: 

CH2OH  +  O  =  CH20  —  H20. 

Formaldehyde  is  a  gaseous  compound  having  the  chemical  formula 
CH2O  and  possessed  of  an  extremely  irritating  odor.  At  a  temperature 
of  68°  F.  the  gas  is  polymerized — that  is  to  say,  a  second  body  is  formed, 


THE  DESTRUCTION  OF  BACTERIA  BY  CHEMICALS  113 

composed  of  a  union  of  two  molecules  of  CH2O.  This  is  known  as  a 
para  formaldehyde,  and  is  a  white,  soapy  body,  soluble  in  boiling  water 
and  alcohol;  it  exists  in  the  solution  of  commerce — a  clear,  watery 
liquid  containing  from  33  to  40  per  cent,  of  the  gas  and  10  to  20  per 
cent,  of  methyl-alcohol,  its  chief  impurity.  If  the  commercial  solution— 
ordinarily  known  in  the  trade  as  ''formalin" — is  evaporated  or  con- 
centrated above  40  per  cent.,  paraformaldehyde  results;  and  when  this 
is  dried  in  vacua  over  sulphuric  acid  a  third  body — trioxymethylene — 
is  produced,  consisting  of  three  molecules  of  CH2O.  This  is  a  white 
powder,  almost  soluble  in  water  or  alcohol,  and  giving  off  a  strong  odor 
of  formaldehyde.  The  solid  polymers  of  formaldehyde,  when  heated, 
are  again  reduced  to  the  gaseous  condition;  ignited,  they  finally  take 
fire  and  burn  with  a  blue  flame,  leaving  but  little  ash. 

Formaldehyde  has  an  active  affinity  for  many  organic  substances, 
and  forms  with  some  of  them  definite  chemical  combinations.  It  com- 
bines readily  with  ammonia  to  produce  a  compound  called  ammoniacal 
aldehyde,  which  possesses  neither  the  odor  nor  the  antiseptic  properties 
of  formaldehyde.  This  action  is  made  use  of  in  neutralizing  the  odor 
of  formaldehyde  when  it  is  desired  to  dispel  it  rapidly  after  disinfection. 
Formaldehyde  also  forms  combinations  with  certain  aniline  colors — 
viz.,  fuchsin  and  safranin — the  shades  of  which  are  thereby  changed 
or  intensified.  These  are  the  only  colors,  however,  which  are  thus 
affected,  and  as  they  are  seldom  used  in  dyeing,  owing  to  their  liability 
to  fade,  this  effect  is  of  little  practical  significance.  The  most  delicate 
fabrics  of  silk,  wool,  cotton,  fur,  leather,  etc.,  are  unaffected  in  texture 
or  color  by  formaldehyde.  Iron  and  steel  are  attacked,  after  long 
exposure,  by  the  gas,  and  more  so  by  its  solution;  but  copper,  brass, 
nickel,  zinc,  silver,  and  gilt  work  are  not  at  all  acted  upon.  Formal- 
dehyde unites  with  nitrogenous  products  of  decay — fermentation  or 
decomposition — forming  true  chemical  compounds,  which  are  odorless 
and  sterile.  It  is  thus  a  true  deodorizer  in  that  it  does  not  replace  one 
odor  by  another  more  powerful,  but  forms  new  chemical  compounds 
which  are  odorless.  Formaldehyde  has  a  peculiar  action  upon  albumin, 
which  it  transforms  into  an  insoluble  and  indecomposable  substance. 
It  renders  gelatin  insoluble  in  boiling  water  and  most  acids  and  alkalies. 
It  is  from  this  property  of  combining  chemically  with  the  albuminoids 
forming  the  protoplasm  of  bacteria  that  formaldehyde  is  supposed  to 
derive  its  bactericidal  powers.  Formaldehyde  is  an  excellent  preserva- 
tive of  organic  products.  It  has  been  proposed  to  make  use  of  this 
action  for  the  preservation  of  meat,  milk,  and  other  food  products; 
but,  according  to  Trillat  and  other  investigators,  formaldehyde  renders 
these  substances  indigestible  and  unfit  for  food.  It  has  been  successively 
employed  as  a  preservative  of  pathological  and  histological  specimens. 

There  are  no  exact  experiments  recorded  of  the  physiological  action 
of  formaldehyde  on  the  human  subject  when  taken  internally.  Slater  and 
Rideal1  report  that  a  1  per  cent,  solution  has  been  taken  in  considerable 

i  Lancet,  April  21, 1894. 


114  PRINCIPLES  OF  BACTERIOLOGY 

quantity  without  serious  results;  and  trioxymethylene  has  been  givenrj 
in  doses  up  to  90  grains  as  an  intestinal  antiseptic.  The  vapors  OM 
formaldehyde  are  extremely  irritating  to  the  mucous  membrane  of  theJ 
eyes,  nose,  and  mouth,  causing  profuse  lacrymation,  coryza,  and  flow* 
of  saliva.  Aronson  reports  that  in  many  of  his  experiments  rabbits* 
and  guinea-pigs  allowed  to  remain  for  twelve  and  twenty-four  hourss 
in  rooms  which  were  being  disinfected  with  formaldehyde  gas  were* 
found  to  be  perfectly  well  when  the  rooms  were  opened.  On  autopsyv 
the  animals  showed  no  injurious  effects  of  the  gas.  Others  have  noticedl 
that  animals,  such  as  dogs  and  cats,  which  have  accidentally  been  con- 
fined for  any  length  of  time  in  rooms  undergoing  formaldehyde  disin- 
fection occasionally  died  from  the  effects  of  the  gas.  Many  observers,, 
however,  have  reported  that  insects,  such  as  roaches,  flies,  and  bed-- 
bugs, are  not,  as  a  rule,  affected.  The  result  of  these  observations  < 
would  seem  to  indicate  that  although  formaldehyde  is  comparatively, 
non-toxic  to  the  higher  forms  of  animal  life,  nevertheless  a  certain  i 
degree  of  caution  should  be  observed  in  the  use  of  this  agent. 

The  results  of    numerous  experiments  have  shown  that  in  the  airi 
2.5  per  cent,  by  volume  of  the  aqueous  solution,  or  1  per  cent,  by  volume • 
of  the  gas,  are  sufficient  to  destroy  fresh  virulent  cultures  of  the  common  i 
pathogenic  bacteria  in  a  few  minutes.     The  researches  of  Pottevin  i 
and  Trillat  have  shown  that  the  germicidal  power  of  the  gas  depends  '> 
not  only  upon  its  concentration,  but  also  upon  the  temperature  and  the 
condition  of  the  objects  to  be  sterilized.     As  with  other  gaseous  disin-' 
fectants — viz.,  sulphur  dioxide  and  chlorine — it  has  been  found  that  the 
action  is  more  rapid  and  complete  at  higher  temperatures — i.  e.,  at  35°j 
to  45°  C.  (95°  to  120°  F.)— and  when  the  test  objects  are  moist  than  at ; 
lower  temperatures  and  when  the  objects  are  dry.     Still,  it  has  been 
repeatedly  demonstrated  by  actual  experiment  in  rooms  that  it  is  pos- 
sible to  disinfect  the  surface  of  apartments  and  articles  contained  in 
them,  under  the   conditions  of   temperature   and   moisture   ordinarily 
existing  in  rooms  even  in  winter,  by  an  exposure  of  a  few  hours  to  a 
saturated  atmosphere  of  formaldehyde  gas. 

Stahl  has  shown  that  bandages   and   iodoform  gauze  can  be  kept 
well   sterilized   by  placing  in   the   jars   containing  them   pieces   of  a] 
preparation   of    paraforrn aldehyde  in   tablet    form   containing  50  per 
cent,  of   formaldehyde.      The  same  experimenter  has    also  succeeded 
in  making  carpets  and  articles  of  clothing  germ-free  by  spraying  them 
with  0.5  to  2  per  cent,  solution  of  formaldehyde  for  fifteen  to  twenty 
minutes  without  the  color  of  the  fabrics  being  in   any  way  affected. 
The   investigations   of   Trillat,    Aronson,    Pottevin,    and    others   have  | 
shown  that  a  concentration  of  yolhro  °f  tne  aqueous  solution  (40  per  j 
cent.),  equal  to  YTTHTO  °f  Pure  formaldehyde,  was  safe  and  sufficiently 
powerful  to  retard  bacterial  growth. 

A  2  per  cent,  watery  solution  of  formalin  destroys  the  vegetative 
forms  of  bacteria  within  five  minutes.  In  our  experiments  formalin 
has  upon  the  vegetative  forms  about  one-half  the  strength  of  pure 
carbolic  acid. 


TIU-:  i)i-:srj<i'CTio\  OF  BACTERIA  BY  CHEMICALS        115 

Chloroform.- This  substance,  even  in  pure  form,  does  not  destroy 

spores,  but  it  does  bacteria  in  vegetative  form,  even  in  1  per  cent,  solu- 

I  tion.     Chloroform  is  used  practically  in  sterilizing  and  keeping  sterile 

j  blood  serum,  which  can  be  used  later  for  culture  purposes  by  driving 

i  off  the  chloroform. 

lodoform. — This  substance  has  but  very  little  destructive  action  upon 
bacteria;  indeed,  upon  most  varieties  it  has  no  appreciable  effect  what- 
ever. When  mixed  with  putrefying  matter,  wround  discharges,  etc., 
the  iodoform  is  reduced  into  soluble  iodine  compounds,  which  partly 
act  destructively  upon  the  bacteria  and  partly  unite  with  the  poisons 
already  produced. 

Carbolic  Acid  (C6H5OH). — A  solution  having  1  part  to  1000  inhibits 
i  the  growth  of  bacteria;  1  part  to  400  kills  the  less  resistant  bacteria, 
and  1  part  to  100  kills  the  remainder.    A  5  per  cent,  solution  kills  the 
less  resistant  spores  within  a  few  hours  and  the  more  resistant  in  from 
one  day  to  four  weeks.    A  slight  increase  in  temperature  aids  the  destruc- 
tive action;  thus,  even  at  37.5°  spores  are  killed  in  three  hours.     A  3 
|  per  cent,  solution  kills  streptococci,  staphylococci,  anthrax  bacilli,  etc., 
within  one  minute.    Carbolic  acid  loses  much  of  its  value  when  in  solu- 
ition  in  alcohol  or  ether.    An  addition  of  0.5  HC1  aids  its  activity.    Car- 
bolic acid  is  so  permanent  and  so  comparatively  little  influenced  by 
'albumin  that  it  is  rightly  widely  used  in  practical  disinfection  even  in 
[places  of  more  powerful  substances. 

Cresol.— Cresol  [CfiH4(CH3)OH]  is  the  chief  ingredient  of  the  so- 
(Called  "crude  carbolic  acid."  This  is  almost  insoluble  in  water,  and 
|  therefore  of  restricted  value.  Many  methods  are  used  for  bringing  it  into 
i  solution  so  as  to  make  use  of  its  powerful  disinfecting  properties.  With 
'equal  parts  of  crude  sulphuric  acid  it  is  a  powerful  disinfectant,  but 
it  is,  of  course,  strongly  corrosive.  An  alkaline  emulsion  of  the  cresols 
jand  other  products  contained  in  "crude"  carbolic  acid  with  soap  is 
I  called  creolin.  It  is  used  in  1  to  5  per  cent,  emulsions.  It  is  fully  as 
[powerful  as  pure  carbolic  acid.  Lysol  is  similar  to  creolin,  except  that 
it  lias  more  of  the  cresols  and  less  of  the  other  products.  It  and  creolin 
pre  of  about  the  same  value. 

Tricresol. — Tricresol  is  a  refined  mixture  of  the  three  cresols  (meta- 
rcresol,  paracresol,  and  orthocresol).  It  is  soluble  in  water  to  the 
extent  of  2.5  per  cent.,  and  is  about  three  times  the  strength  of  carbolic 
acid. 

Aniline  Dyes. — Some  of  these  colors  possess  marked  germicidal 
[qualities.  According  to  observers,  methyl  violet  (pyoktanin)  and 
(malachite  green  destroy  the  typhoid  bacillus  in  bouillon  cultures  in 
jthe  proportion  of  1 : 200  in  two  hours'  exposure,  and  the  pyogenic  cocci 
in  less.  In  1: 100,000  solutions  they  are  said  to  retard  the  development 
[of  bacteria. 

Oil  of  turpentine,  1 :  200,  prevents  the  growth  of  bacteria. 
Camphor  has  very  slight  antiseptic  action. 

(Yen-ore  in  1:200  kills  many  bacteria  in  ten  minutes;  1:100  failed 
to  kill  tubercle  bacilli  in  twelve  hour-. 


116 


PRINCIPLES  OF  BACTERIOLOGY 


Essential  oils :  Cardeac  and  Meumir  found  that  the  essences  of  cin- 
namon, cloves,  thyme,  and  others  killed  typhoid  bacilli  within  one 
hour.  Sandalwood  required  twelve  hours. 

Thymol  and  eucalyptol  have  about  one-fourth  the  strength  of  car- 
bolic acid  (Behring). 

Oil  of  peppermint  in  1 : 100  solution  prevents  the  growth  of  bacteria. 


Aluminum  acetate     . 

.    1     6000 

Ammonium  chloride. 

1 

9 

Boric  acid    . 

.    1 

143 

Calcium  chloride 

.    1 

25 

Calcium  hypochlorite 

.    1 

1000 

Carbolic  acid 

.    1 

333 

Chloral  hydrate  . 

.    1 

107 

Cupric  sulphate  . 

.    1 

2000 

Ferrous  sulphate 

.    1 

200 

Formaldehyde  (40  %). 

.    1 

10,000 

Hydrogen  peroxide   . 

.    1  :  20,000 

TABLE  OF  ANTISEPTIC  VALUES.1 


Mercuric  chloride 
Mercuric  iodide  . 
Potassium  bromide    . 
Potassium  iodide 
Potassium  permanganate 
Pure  formaldehyde    . 
Quinine  sulphate 
Silver  nitrate      . 
Sodium  borate    .  '     . 
Sodium  chloride 
Zinc  chloride 
Zinc  sulphate 


14,300 

40,000 

10 

10 

300 

25,000 

800 

12,500 

14 

6 

500 

20 


1  These  figures  are  approximately  correct,  and  represent  the  percentage  of  disinfectant  required 
to  be  added  to  a  fluid  containing  considerable  organic  material,  in  order  to  permanently  inhibit  any 
bacterial  growth . 


CHAPTER  X. 

PRACTICAL  DISINFECTION  AND  STERILIZATION  (HOUSE,  PERSON, 

INSTRUMENTS,  AND  FOOD)— STERILIZATION  OF  MILK 

FOR  FEEDING  INFANTS. 

Disinfectants  and  Methods  of  Disinfection  Employed  in  the 
House  and  Sick-room. 

Disinfection  and  Disinfectants. — Sunlight,  pure  air,  and  cleanliness 
are  always  very  important  agents  in  maintaining  health  and  in  protect- 
ing the  body  against  many  forms  of  illness.  When,  however,  it  becomes 
necessary  to  guard  against  such  special  dangers  as  accumulated  filth 
or  contagious  diseases,  disinfection  is  essential.  In  order  that  disin- 
fection shall  afford  complete  protection  it  must  be  thorough;  and  per- 
fect cleanliness  is  better,  even  in  the  presence  of  contagious  disease, 
than  filth  with  poor  disinfection. 

Since  all  forms  of  fermentation,  decomposition,  and  putrefaction, 
as  well  as  the  infectious  and  contagious  diseases,  are  caused  by  micro- 
organisms, it  is  the  object  of  disinfection  to  kill  these.  Decomposition 
and  putrefaction  should  at  all  times  be  prevented  by  the  immediate 
destruction  or  removal  from  the  neighborhood  of  the  dwelling  of  all 
useless  putrescible  substances.  In  order  that  as  few  articles  as  possible 
shall  be  exposed  to  the  germs  causing  the  contagious  diseases,  and  thus 
become  carriers  of  infection,  it  is  important  that  all  articles  not  neces- 
sary for  immediate  use  in  the  care  of  the  sick  person,  especially  uphol- 
stered furniture,  carpets,  and  curtains,  should  be  removed  from  the 
room  before  placing  the  sick  person  in  it. 

Agents  for  Cleansing  and  Disinfection. 

Too  much  emphasis  cannot  be  placed  upon  the  importance  of  cleanli- 
ness, both  as  regards  the  person  and  the  dwelling,  in  preserving  health 
and  protecting  the  body  from  all  kinds  of  infectious  disease.  Sunlight 
and  fresh  air  should  be  freely  admitted  through  open  windows,  and 
personal  cleanliness  should  be  attained  by  frequently  washing  the 
hands  and  body. 

Cleanliness  in  dwellings,  and  in  all  places  where  men  go,  may,  under 
ordinary  circumstances,  be  well  maintained  by  the  use  of  the  two  fol- 
lowing solutions: 

1.  Soapsuds  Solution. — For  simple  cleansing,  or  for  cleansing  after 
the  method  of  disinfection  by  chemicals  described  below,  one  ounce  of 
common  soda  should  be  added  to  twelve  quarts  of  hot  soapsuds  (soft 
soap  and  water). 


118  PRINCIPLES  OF  BACTERIOLOGY 


2.  Strong  Soda  Solution. — This,  which  is  a  stronger  and  more  effective 
cleansing  solution  and  also  a  feeble  disinfectant,  is  made  by  dissolving 
one-half  pound  of  common  soda  in  three  gallons,  of  hot  water.     The 
solution  thus  obtained  should  be  applied  by  scrubbing  with  a  hard  brush. 

When  it  becomes  necessary  to  arrest  putrefaction  or  to  prevent  the 
spread  of  contagious  diseases  by  surely  killing  the  living  germs  which 
cause  them,  more  powerful  agents  must  be  employed  than  those  re- 
quired for  simple  cleanliness,  and  these  are  commonly  called  disin- 
fectants. The  following  are  some  of  the  most  reliable  ones : 

3.  Heat. — Complete  destruction  by  fire  is  an  absolutely  safe  method 
of  disposing  of  infected  articles  of  small  value,  but  continued  high 
temperatures  not  as  great  as  that  of  fire  will  destroy  all  forms  of  life; 
thus,  boiling  or  steaming  in  closed  vessels  for  one-half  hour  will  abso- 
lutely destroy  all  disease  germs. 

4.  Carbolic  Acid  Solution. — Dissolve  six  ounces  of  carbolic  acid  in 
one  gallon  of  hot  water.    This  makes  approximately  a  5  per  cent,  solu- 
tion of  carbolic  acid,  which,  for  many  purposes,  may  be  diluted  with 
an  equal  quantity  of  water.     The  commercial  ''soluble  crude  carbolic 
acid  "  can  be  used  instead  of   the  pure  carbolic  acid  for  privies  and 
drains.     It   makes   a   white   emulsion    on  account  of  its  not  entering 
readily  into    solution.     Care    must  be  taken  that  the  pure  acid  does 
not  come  in  contact  with  the  skin. 

5.  Bichloride  Solution  (bichloride  of  mercury  or  corrosive  sublimate). 
—Dissolve  sixty  grains  of  pulverized  corrosive  sublimate  and  two  table- 
spoonfuls  of  common  salt  in  one  gallon  of  hot  water.     This  solution, 
which  is  approximately   1:1000,  must   be  kept  in  glass,  earthen,  or 
wooden  vessels  (not  in  metal  vessels).     For  safety  it  is  well  to  cover 
the  solution. 

The  carbolic  and  bichloride  solutions  are  very  poisonous  when  taken 
by  the  mouth,  but  are  harmless  when  used  externally. 

6.  Milk  of  Lime. — This  mixture  is  made  by  adding  one  quart  of  dry, 
freshly  slaked  lime  to  four  or  five  quarts  of  water.     (Lime  is  slaked  by 
pouring  a  small  quantity  of  water  on  a  lump  of  quicklime.    The  lime 
becomes  hot,  crumbles,  and  as  the  slaking  is  completed  a  white  powder 
results.     The  powder  is  used  to  make  milk  of  lime.)     Air-slaked  lime 
(the  carbonate)  has  no  value  as  a  disinfectant. 

7.  Dry  "Chloride  of  Lime."— This  must  be  fresh  and  kept  in  closed 
vessels  or  packages.    It  should  have  the  strong,  pungent  odor  of  chlorine. 

.  Formalin.— Add  1  part  of  formalin  to  10  of  water.  This  equals 
in  value  the  5  per  cent,  carbolic  acid  solution. 

9.  Creolin,  tricresol,  and  lysof  are  of  about  the  same  value  as  pure 
carbolic  acid. 

The  proprietary  disinfectants,  which  are  so  often  widely  advertised 
and  whose  composition  is  kept  secret,  are  relatively  expensive  and 
often  unreliable  and  inefficient.  It  is  important  to  remember  that 
substances  which  destroy  or  disguise  bad  odors  are  not  necessarily 
disinfectants,  and  that  there  are  very  few  disinfectants  that  are  not 
poisonous  when  taken  internally. 


PRACTICAL  DISINFECTION  AM)  STERILIZATION  119 

[NOTE. — The  cost  of  the  pure  carbolic  acid  solution  is  much  greater 
than  that  of  most  of  the  other  solutions,  but  except  for  the  disinfection 
of  the  skin,  which  in  some  persons  it  irritates,  and  of  woodwork,  it  is 
generally  much  to  be  preferred  by  those  not  thoroughly  familiar  with 
disinfectants,  as  it  does  not  deteriorate,  and  is  rather  more  uniform  in 
its  action  than  some  of  the  other  disinfectants.! 


Methods  of  Disinfection  in  Infectious  and  Contagious  Diseases. 

The  diseases  to  be  commonly  guarded  against,  outside  of  surgery, 
by  disinfection  are  scarlet  fever,  measles,  diphtheria,  tuberculosis, 
smallpox,  typhoid  and  typhus  fever,  bubonic  plague  and  cholera. 

1.  Hands  and   Person. — Dilute  the  carbolic  solution  with  an  equal 
amount  of  water  or  use  the  bichloride  solution  without  dilution.    Hands 
soiled  in  caring  for  persons  suffering  from  contagious  diseases,  or  soiled 
portions  of  the  patient's  body,  should  be  immediately  and  thoroughly 
washed  with  one  of  these  solutions  and  then  washed  with  soap  and 
water,  and  finally  immersed  again  in  the  solutions.     The  nails  should 
always  be  kept  perfectly  clean.     Before  eating,  the  hands  should  be 
first  washed  in  one  of  the  above  solutions,  and  then  thoroughly  scrubbed 
with  soap  and  water  by  means  of  a  brush. 

2.  Soiled    clothing,   towels,  napkins,  bedding,  etc.,  should    be    imme- 
diately immersed  in  the  carbolic  solution,  in  the  sick-room,  and  soaked 
for  one  or  more  hours.    They  should  then  be  wrung  out  and  boiled  in 
the  soapsuds  solution  for  one  hour.     Articles  such  as  beds,  woollen 
clothing,  etc.,  which  cannot  be  washed,  should  at  the  end  of  the  disease 
be  referred  to  the  Health  Department,  if  such  is  within  reach,  for  dis- 
infection or  destruction;  or  if  there  is  no  public  disinfection,  these  goods 
should  be  thoroughly  exposed  to  formaldehyde  gas,  as  noted  later. 

3.  Food  and  Drink. — Food  thoroughly  cooked  and  drinks  that  have 
been  boiled  are  free  from  disease  germs.    Food  and  drinks,  after  cook- 
ing or  boiling,  if  not  immediately  used,  should  be  placed  when  cool  in 
clean  dishes  or  vessels  and  covered.     In  the  presence  of  an  epidemic 
of  cholera  or  typhoid  fever,  milk  and  water  used  for  drinking,  cooking, 
washing  dishes,  etc.,  should   be  boiled  before  using,  and  all  persons 
should  avoid  eating  uncooked  fruit  and  fresh  vegetables.     Instead  of 
boiling  milk  may  be  heated  to  80°  C.  for  one-half  hour. 

4.  Discharges  of   all  kinds  from  the  mouth,  nose,  bladder,  and  bowels 
of  patients  suffering  from  contagious  diseases  should  be  received  into 
glass  or  earthen  vessels  containing  the  carbolic  solution,  or  milk  of  lime, 
or  they  should  be  removed  on  pieces  of  cloth,  which  are  immediately 
immersed  in  one  of  these  solutions.     Special  care  should  be  observed 
to  disinfect  at  once  the  vomited  matter  and  the  intestinal  discharges 
from  cholera  patients.     In  typhoid  fever  the  urine  and  the  intestinal 
discharges,  and  in  diphtheria,  measles,  and  scarlet  fever  the  discharges 
from  the  throat  and  nose  all  carry  infection  and  should  be  treated  in 
the  same  manner.     The  volume  of  the  solution  used  to  disinfect  dis- 
charges should  be  at  least  twice  as  great  as  that  of  the  discharge,  and 


120  PRINCIPLES  OF  BACTERIOLOGY 

should  completely  mix  with  and  cover  it.  After  standing  for  an  hour 
or  more  the  disinfecting  solution  with  the  discharges  may  be  thrown 
into  the  water-closet.  Cloths,  towels,  napkins,  bedding,  or  clothing 
soiled  by  the  discharges  must  be  at  once  placed  in  the  carbolic  solution, 
and  the  hands  of  the  attendants  disinfected,  as  described  above.  In 
convalescence  from  measles  and  scarlet  fever  the  scales  from  the  skin 
are  also  carriers  of  infection.  To  prevent  the  dissemination  of  disease 
by  means  of  these  scales  the  skin  should  be  carefully  washed  daily  in 
warm  soap  and  water.  After  use  the  soapsuds  should  be  disinfected 
and  thrown  into  the  water-closet. 

Masses  of  feces  are  extremely  difficult  to  disinfect  except  on  the 
surface,  for  it  takes  disinfectants  such  as  the  carbolic  acid  solution 
some  twelve  hours  to  penetrate  to  their  interior.  If  fecal  masses  are  to 
be  thrown  into  places  where  the  disinfectant  solution  covering  them 
will  be  washed  off,  it  will  be  necessary  to  be  certain  that  the  disinfectant 
has  previously  penetrated  to  all  portions  and  destroyed  the  disease 
germs.  This  can  be  brought  about  by  stirring  them  with  the  disin- 
fectant and  allowing  the  mixture  to  stand  for  one  hour,  or  by  washing 
them  into  a  pot  holding  soda  solution  which  is  already  at  the  boiling 
temperature,  or  later  will  be  brought  to  one. 

5.  Sputum  from  Consumptive  Patients. — The  importance  of  the  proper 
disinfection  of  the  sputum  from  consumptive  patients  is  still  under- 
estimated.     Consumption  is  an  infectious   disease,  and  is  always   the 
result  of  transmission  from  the  sick  to  the  healthy  or  from  animals  to 
man.    The  sputum  contains  the  germs  which   cause  the  disease,  and 
in  a  large  proportion  of  cases  is  the  source  of  infection.     After  being 
discharged,  unless  properly  disposed  of,  it  may  become  dry  and  pul- 
verized and  float  in  the  air  as  dust.    This  dust  contains  the  germs,  and 
is  a  common  cause  of  the  disease,  through  inhalation.     In  all  cases, 
therefore,  the  sputum  should  be  disinfected  when  discharged.    It  should 
be  received    in  covered    cups  containing  the  carbolic  or  milk-of-lime 
solution.     Handkerchiefs  soiled  by  it  should  be  soaked  in  the  carbolic 
solution  and  then  boiled.     Dust  from  the  walls,  mouldings,  pictures, 
etc.,  in  rooms  that  have  been  occupied  by  consumptive  patients,  where 
the  rules  of  cleanliness  have  not  been  carried  out,  contain  the  germs 
and  will  produce  tuberculosis  in  animals  when  used  for  their  inoculation; 
therefore,  rooms  should  be  thoroughly  disinfected  before  they  are  again 
occupied.     If  the  sputum  of  all  consumptive  patients  were  destroyed 
at  once  when  discharged  a  large  proportion  of  the  cases  of  the  disease 
would  be  prevented. 

6.  Closets,  Kitchen  and  Hallway  Sinks,  etc. — The  closet  should  never 
be  used  for  infected  discharges  until  they  have  been  thoroughly  disin- 
fected, if  it  can  be  avoided;  if  done,  one  pint  of  carbolic  solution  should 
be  poured  into  the  pan  (after  it  is  emptied)  and  allowed  to  remain  there. 
Sinks  should  be  flushed  at  least  once  daily. 

7.  Dishes,  knives,  forks,  spoons,  etc.,  used   by  a  patient  should,  as  a 
rule,  be  kept  for  his  exclusive  use  and  not  removed  from  the  room. 
They  should  be  washed  first  in  the  carbolic  solution,  then  in  boiling 


PRACTICAL  DISINFECTION  AND  STERILIZATION  121 

hot  soapsuds,  and  finally  rinsed  in  hot  water.  These  washing  fluids 
should  afterward  be  thrown  into  the  water-closet.  The  remains  of 
the  patient's  meals  may  be  burned  or  thrown  into  a  vessel  containing 
the  carbolic  solution  or  milk  of  lime,  and  allowed  to  stand  for  one  hour 
before  being  thrown  away. 

8.  Rooms  and  Their  Contents. — Rooms  which  have  been  occupied  by 
persons  suffering  from  contagious  disease  should  not  be  again  occupied 
until  they  have  been  thoroughly  disinfected.     For  this  purpose  either 
careful  fumigation  with  formaldehyde  gas  or  sulphur  should  be  em- 
ployed, or  this  combined  with  the  following  procedure:    Carpets,  cur- 
tains, and  upholstered  furniture  which  have  been  soiled  by  discharges, 
or  which  have  been  exposed  to  infection  in  the  room  during  the  illness, 
will  be  removed  for  disinfection  to  chambers  where  they  can  be  exposed 
to  formaldehyde  gas  and  moderate  warmth  for  twelve  to  twenty-four 
hours,  or  to  steam.     Some  carpets,  such  as  many  Wiltons,  are  dis- 
colored by  moist  steam.    These  must  be  put  in  the  formaldehyde  cham- 
ber.    Woodwork,  floors,  and  plain  furniture  will  be  thoroughly  washed 
with  the  soapsuds  and  bichloride  solutions. 

9.  Rags,  cloths,  and  articles  of  small  value,  which   have   been   soiled 
by  discharges  or  infected  in  other  ways,  should  be  boiled  or  burned. 

10.  In  case  of  death  the  body  should    be    completely  wrapped    in 
several  thicknesses  of  cloth  wrung  out  of  the  carbolic  or  bichloride 
solution,  and  when  possible  placed  in  an  hermetically  sealed  coffin. 

It  is  important  to  remember  that  an  abundance  of  fresh  air,  sunlight, 
and  absolute  cleanliness  not  only  helps  protect  the  attendants  from 
infection  and  aid  in  the  recovery  of  the  sick,  but  directly  destroys  the 
bacteria  which  cause  disease. 

Methods   of   Cleanliness  and  Disinfection  to  Prevent  the 
Occurrence  of  Illness. 

1.  Water-closet  bowls  and  all  receptacles  for  human  excrement  should 
be  kept  perfectly  clean  by  frequent  flushing  with  a  large  quantity  of 
water,  and  as  often  as  necessary  disinfected  with  the  carbolic,  bichloride, 
or  other  efficient  solutions.    The  woodwork  around  and  beneath  them 
should  be  frequently  scrubbed  with  the  hot  soapsuds  solution. 

2.  Sinks  and  the  woodwork  around  and  the  floor  beneath  them  should 
be  frequently  and  thoroughly  scrubbed  with  the  hot  soapsuds  solution. 

3.  School  Sinks. — School  sinks  should  be  thoroughly  flushed  with  a 
large  quantity  of  water  at  least  twice  daily,  and  should  be  carefully 
cleaned  twice  a  week  or  oftener  by  scrubbing.     Several  quarts  of  the 
carbolic  solution  should  be  frequently  thrown  in  the  sink  after  it  has 
been  flushed. 

4.  Cesspools  and  Privy  Vaults. — An  abundance  of  milk  of  lime  or 
chloride  of  lime  should  be  thrown  into  these  daily,  and  their  contents 
should  be  frequently  removed. 

5.  Cellars  and  rooms  in  cellars  are  to  be  frequently  whitewashed,  and, 
if  necessary,  the  floors  sprinkled  with  dry  chloride  of  lime.    Areas  and 


122  PRINCIPLES  OF  BACTERIOLOGY 

paved  yards  should  be  cleaned,  scrubbed,  and,  if  necessary,  washed 
with  the  bichloride  solution.  Street  gutters  and  drains  should  be  cleaned 
and,  when  necessary,  sprinkled  with  chloride  of  lime  or  washed  with 
milk  of  lime. 

6.  Air-shafts. — Air-shafts  should  be  first  cleaned  thoroughly  and  then 
whitewashed.     To  prevent  tenants  throwing  garbage  down  air-shafts 
it  is  sometimes  advisable  to  put  wire  netting  outside  of  windows  open- 
ing on  shafts.    Concrete  or  asphalt  bottoms  of  shafts  should  be  cleaned 
and  washed  with  the  bichloride  solution  or  sprinkled  with  chloride  of 
lime. 

7.  Hydrant    sinks,  garbage   receptacles,  and    garbage   and    oyster-shell 
chutes  and  receptacles  should  be  cleaned  daily  and  sprinkled  with  dry 
chloride  of  lime. 

8.  Refrigerators  and   the   surfaces   around    and    beneath  them,  dumb- 
waiters, etc.,  may  be  cleaned  by  scrubbing  them  with  the  hot  soapsuds 
solution. 

9.  Traps. — All  traps  should  be  flushed  daily  with  an  abundance  of 
water.    If  at  any  time  they  become  foul  they  may  be  cleaned  by  pouring 
considerable  quantities  of  the  hot  strong  soda  solution  into  them,  fol- 
lowed by  the  carbolic  solution. 

10.  Urinals  and  the  floors  around  and  beneath  them  should  be  cleaned 
twice  daily  with  the  hot  soapsuds  solution,  and  in  addition  to  this,  if 
offensive,  they  may  be  disinfected  with  the  carbolic  solution. 

11.  Stable  Floors  and  Manure  Vaults. — Stable  floors  should  be  kept 
clean  and  occasionally  washed  with  the  hot  soapsuds  or  the  hot  strong 
soda  solution.     Powdered  fresh  chloride  of  lime  or  formalin  may  be 
used  in  manure  vaults. 

12.  Vacant  rooms  should  be  frequently  aired. 

13.  The  woodwork  in  school-houses  should  be  scrubbed  weekly  with  hot 
soapsuds.    This  refers  to  floors,  doors,  door-handles,  and  all  woodwork 
touched  by  the  scholars'  hands. 

14.  Spittoons  in  all  public  places  should  be  emptied  daily  and  washed 
with  the  hot  soapsuds  solution,  after  which  a  small  quantity  of  the  car- 
bolic solution  or  milk  of  lime  should  be  put  in  the  vessel  to  receive  the 
expectoration. 

15.  Cars,    Ferry-boats,    and    Public    Conveyances. — The    floors,    door- 
handles, railings,  and  all  parts  touched  by  the  hands  of  passengers 
should  be  washed  frequently  with  the  hot  soapsuds  solution.    Slat-mats 
from  cars,  etc.,  should  be  cleaned  by  scrubbing  with  a  stiff  brush  in 
the  hot  soapsuds  solution. 

Telephone  receiver  mouth-pieces  should  also  be  frequently  cleansed. 

Use  of  Bromine  Solution  as  a  Deodorant. — Slaughter-houses,  butchers' 
ice-boxes  and  wagons,  trenches,  excavations,  stable  floors,  manure-vaults, 
dead  animals,  offal,  offal  docks,  etc.,  may  be  deodorized  by  a  weak  solu- 
tion of  bromine,  which  is  a  valuable  agent  for  this  purpose.  The  bromine 
solution,  however,  is  only  temporary  in  its  action,  and  must  be  used 
repeatedly.  It  should  be  applied  by  sprinkling.  Although  somewhat 
corrosive  in  its  action  on  metals,  it  is  otherwise  harmless. 


PRACTICAL  DISINFECTION  AND  STK,;U.I'/..\  TION  123 

The  solution  of  bromine  must  be  prepared  with  great  care,  as  the 
pure  bromine  from  which  it  is  made  is  dangerous.  It  is  very  caustic 
when  brought  in  contact  with  the  skin;  it  is  volatile  and  its  fumes  are 
very  irritating  when  inhaled.  To  prepare  the  solution  an  ounce  bottle 
of  liquid  bromine  is  dropped  into  three  gallons  of  water,  and  broken 
under  the  water  and  thoroughly  stirred. 

The  Practical  Employment  of  Formaldehyde  and  Sulphur  Dioxide  Gases 
in  the  Surface  Disinfection  of  Rooms  and  the  Disinfection  of  Goods 
which  would  be  Injured  by  Heat. — Formaldehyde  gas  has  come  into 
such  general  use,  and  is  for  many  purposes  so  valuable,  that  the 
description  of  methods  employed  to  generate  and  use  it  will  be  given  in 
detail. 

If  we  consider  now  the  practical  application  of  formaldehyde  gas 
for  purposes  of  disinfection  we  find  that  its  destructive  action  on  micro- 
organisms depends  upon  a  number  of  factors,  the  chief  of  which  are  its 
concentration  in  the  surrounding  atmosphere,  the  length  of  the  con- 
tact, the  existing  temperature,  the  accompanying  moisture,  and  the 
nature  of  the  organism. 

The  necessary  concentration  of  the  gas  in  the  surrounding  atmos- 
phere to  kill  the  micro-organisms  varies  with  each  species,  for  some 
resist  chemical  agents  much  more  than  others,  and  also  with  the  freedom 
of  access  of  the  gas  to  the  bacteria,  for  if  they  are  under  cover  or  within 
fabrics  a  greater  amount  of  gas  must  be  generated  than  if  they  are  freely 
exposed. 

For  purely  surface  disinfection,  when  the  less  resistant  bacteria  or 
other  micro-organisms  are  to  be  destroyed,  there  will  be  required, 
according  to  the  method  used,  six  to  ten  ounces  of  formalin  of  full 
strength,  or  its  equivalent,  to  1000  cubic  feet. 

For  the  destruction  of  the  more  resistant  but  non-spore  bearing 
forms,  such  as  typhoid  fever  or  tubercle  bacilli,  at  least  twelve  ounces 
of  formalin  should  be  used.  The  gas  penetrates  through  fabrics  with 
difficulty,  and  to  pass  through  heavy  goods  the  concentration  of  the  gas 
must  be  doubled  and  moderate  heat  added  (45  C.°  or  above). 

Value  of  Moisture. — At  first  it  was  thought  that  formaldehyde  gas 
acted  more  effectually  in  a  dry  atmosphere,  but  further  investigation 
has  proved  that,  although  it  does  destroy  bacteria  with  the  amount  of 
moisture  usually  present  in  the  air,  and  contained  in  their  own  sub- 
stance, it  acts  much  more  powerfully  and  certainly  when  additional 
moisture  is  present,  and  best  when  present  up  to  the  point  of  saturation. 
The  actual  spraying  of  walls  and  goods  to  be  disinfected  with  water  is 
even  more  efficacious. 

A  fairly  high  temperature — but  one  still  below  that  which  would 
injure  delicate  fabrics — increases  not  only  the  activity  of  formaldehyde 
gas  but  also  its  penetrative  power,  and  for  heavy  goods  it  is  essential. 
The  production  of  a  partial  vacuum  in  the  chambers  before  the  intro- 
duction of  the  folmaldehyde  gas  still  further  assists  its  penetration. 

The  length  of  exposure  necessary  for  complete  disinfection  depends 
upon  the  nature  of  the  disease  for  which  it  is  carried  out — the  penetra- 

or  THF 
I  iMi\/r  DO  i  T\J 


124  PRINCIPLES  OF  BACTERIOLOGY 

tion  required,  the  concentration  of  the  gas  used,  the  amount  of  moisture 
in  the  air,  the  temperature  of  the  air,  and  the  size  and  shape  of  the 
room.  For  surface  disinfection  in  rooms,  when  as  much  as  twelve 
ounces  of  formalin  are  used  for  each  1000  cubic  feet,  five  hours'  exposure 
is  amply  sufficient,  most  bacteria  being  killed  within  the  first  few  minutes. 
For  the  destruction  of  micro-organisms  protected  by  even  a  layer  of 
thin  covering,  double  the  formalin  and  double  the  time  of  exposure 
should  be  allowed,  and  even  then  the  killing  of  many  species  of  non- 
spore  bearing  bacteria  cannot  be  counted  upon  in  ordinary  rooms. 
When  absolutely  complete  disinfection  is  demanded,  where  penetration 
of  gas  is  required,  the  goods  must  be  placed  in  chambers  where  moderate 
heat  can  be  added  and  all  leakage  of  gas  prevented. 

Various  forms  of  apparatus  can  be  properly  employed  to  liberate 
formaldehyde  gas  for  purposes  of  disinfection.  There  are  two  essentials 
to  any  good  method — namely,  that  the  formaldehyde  gas  is  given  off 
quickly,  and  that  there  is  no  great  loss  by  deterioration  of  the  formalin. 

Wood  Alcohol. — A  number  of  lamps  have  been  devised,  all  very  much 
on  the  same  principle,  though  varying  somewhat  in  mechanical  con- 
struction, which  bring  about  the  incomplete  oxidation  of  methyl-alcohol 
by  passing  the  vapors  mixed  with  air  over  the  incandescent  metal. 
Although  disinfection  can  be  carried  out  by  the  best  of  these  lamps,  in 
our  experience  none  of  them  up  to  the  present  time  are  satisfactory  or 
economical.  They  may  be  very  useful  as  deodorizers  in  the  sick-room 
or  other  places. 

The  same  principle  is  used  efficiently  in  another  form.  The  vapor 
of  wood  alcohol  is  passed  over  surfaces  of  asbestos  containing  particles 
of  finely  divided  platinum.  This  apparatus  has  given  very  good  results, 
and  for  a  given  amount  of  disinfection  leaves  less  odor  of  formaldehyde 
gas  in  the  room  than  any  other.  The  apparatus  is,  however,  bulky  and 
expensive. 

Formochloral  by  the  Trillat  System. — This  system  consists  in  heating, 
under  three  atmospheres  of  pressure,  a  solution  of  formaldehyde  gas 
in  water  mixed  with  30  per  cent,  of  calcium  chloride,  known  as  "formo- 
chloral," to  a  temperature  of  135°  C.  (275°  F.).  It  is  claimed  for  this 
method  of  producing  the  gas  from  formochloral  that  the  polymerization 
of  the  formaldehyde  is  prevented,  which  would  otherwise  take  place 
if  a  solution  of  formaldehyde  were  evaporated  under  ordinary  condi- 
tions, and  that  thereby  the  whole  of  the  formaldehyde  is  obtained  in 
the  gaseous  state.  The  addition  of  any  neutral  salt  aids  the  process, 
it  is  said,  but  calcium  chloride  is  the  best.  The  results  with  this  apparatus 
have  been  satisfactory,  but  not  more  so  than  by  other  methods.  The 
apparatus  is  expensive  and  heavy  and  therefore  unnecessary. 

Formalin  by  Boiling  and  Passing  the  Vapor  through  a  Superheated  Coil 
or  Chamber. — This  system  consists  in  heating  the  ordinary  commercial 
formalin  to  a  temperature  of  about  260°  C.  (500°  F.)  in  an  incandescent 
copper  coil  or  chamber,  and  allowing  the  vapors  to  pass  off  freely.  It  is 
claimed  for  this  method  that  the  degree  of  heat  necessary  to  break 
up  the  polymerized  products  formed  is  supplied,  and  thus  a  loss  of 


PRACTICAL  DISIXFECTIOX  AND  STERILIZATION 


125 


FIG.  65 


formaldehyde  is  prevented.  A  further  action  of  the  intense  heat  in  the 
copper  tube  on  the  solution  is  to  partially  convert  the  methyl-alcohol 
contained  in  commercial  formalin  into  formaldehyde  gas  by  partial 
oxidation,  thereby  preventing  the  formation  of  methyl  and  increasing 
the  amount  of  formaldehyde. 

The  apparatus  consists  of  a  closed  receiver  of  copper  holding  about 
a  gallon,  a  coil  of  copper  pipe  attached  at  one  end  to  the  bottom  of  the 
receiver,  and,  like  the  preceding  apparatus  and  that  made  by  Lentz, 
at  the  other,  by  means  of  a  suitable  connection  (rubber  tube  with  gutta- 
percha  or  metallic  mouth-piece),  with  the  room  or  apartment  to  be 
disinfected,  and  a  heating  lamp  (Swedish  lamp  or  Bunsen  burner).  In 
operation  the  desired  quantity  of  formalin 
is  placed  in  the  receiver  and  the  receiver  is 
closed.  The  lamp  is  lighted  and  the  coil 
brought  to  a  red  heat.  The  valve  is  then 
opened  and  the  solution  contained  in  the 
receiver  is  allowed  to  pass  down  and  into  Q= 
the  coil  in  a  fine  stream.  Upon  coming  in 
contact  with  the  heated  metal  the  formal- 
dehyde solution  is  instantly  decomposed, 
and  the  liberated  gas  is  further  purified  as 
it  progresses  through  the  incandescent  coil. 
The  apparatus  is  liable  to  get  out  of  order, 
in  that  the  valve  is  apt  to  become  clogged 
and  so  stop  the  flow  of  formalin  until  freed 
by  a  wire  supplied  for  the  purpose. 

In  the  new  form  (Fig.  65)  the  formalin 
is  first  boiled  in  the  large  chamber  and 
passes  as  vapor  through  the  tube  connect- 
ing B  and  C.  In  C  it  is  superheated  and 
passes  out  the  tube  D  into  the  room.  In' 
this  apparatus  there  is  nothing  to  get  out 
of  order,  and  it  operates  quickly.  Up  to 
the  present  time  this  is  the  most  practical 
apparatus  we  have  met  with,  when  the  initial  cost,  about  $25,  is  not 
an  objection.  In  all  forms  of  apparatus  where  formalin  is  used  the  large 
receiving  chamber  should  be  washed  out  from  time  to  time  with  hot 
water,  to  remove  any  deposit  there  may  be. 

Trioxymethylene  by  Schering's  System. — This  system  consists  in  heat- 
ing the  solid  polymer  of  formaldehyde  (trioxymethylene)  in  a  lamp 
specially  constructed  for  the  purpose  by  the  Chemische  Fabrik  auf 
Actien,  in  Berlin.  The  trioxymethylene  is  used  in  the  form  of  com- 
pressed tablets  or  pastilles,  as  being  more  convenient  for  use.  Each 
pastille  contains  the  equivalent  of  100  per  cent,  of  formaldehyde  gas, 
according  to  the  manufacturers,  and  weighs  1  gram. 

The  mode  of  using  the  apparatus  is  very  simple:  The  disinfector  is 
placed  upon  a  sheet  of  iron  on  the  floor  of  the  room  to  be  disinfected. 
From  100  to  250  pastilles  can  be  evaporated  at  a  time  in  the  apparatus. 


Formaldehyde  apparatus. 


PRINCIPLES  OF  BACTERIOLOGY 

For  the  production  of  greater  quantities  of  formaldehyde  vapor  several 
of  these  outfits  may  be  used  together.  The  lamp  is  filled  with  ordinary 
or  wood  alcohol,  about  twice  as  many  cubic  centimetres  of  the  alcohol 
being  employed  as  there  are  pastilles  to  be  evaporated.  The  wicks 
should  project  but  little  above  the  necks  of  the  burners,  or  the  apparatus 
may  get  too  hot  and  ignite  the  pastilles.  The  vessel  is  charged  with 
formalin  pastilles  and  the  disinfector  placed  over  the  lighted  spirit  lamp. 
The  lamp  is  then  allowed  to  burn  out  in  the  closed  room.  One  hun- 
dred pastilles  are  considered  to  be  sufficient  for  the  disinfection  of  1000 
cubic  feet  of  space.  Lately,  a  small  steam  boiler  has  been  added  to 
the  apparatus,  for  the  purpose  of  furnishing  sufficient  moisture  with 
the  gas.  The  results  obtained  by  us  in  superficial  disinfection,  when 
from  150  to  200  pastilles  have  been  used  to  each  1000  cubic  feet,  have 
been  good.  The  great  advantage  of  the  method  is  in  the  small  cost  of 
the  apparatus,  $3.00,  and  the  avoidance  of  the  danger  of  deterioration, 
which  is  present  to  some  extent  in  formalin.  Smaller  lamps  are  very 
useful  for  the  deodorization  of  rooms. 

From,  Pastilles  Composed  of  a  Top  of  Compressed  Paraform  and  a 
Base  of  Prepared  Charcoal. — This  is  a  very  neat  but  somewhat  expen- 
sive method  of  liberating  formaldehyde  gas.  Our  results  with  it  have 
been  good. 

Formalin  to  which  Glycerin  has  been  Added. — To  the  formalin  is  added 
10  per  cent,  of  glycerin,  and  the  mixture  is  simply  boiled  in  a  suitable 
copper  vessel,  the  steam  and  formaldehyde  gas  passing  off  by  a  tube. 
This  is  a  very  serviceable  apparatus.  When  it  is  attempted  to  vaporize 
the  formalin  too  rapidly  part  of  it  passes  over  in  fluid  form,  and  is  thus 
wasted. 

With  a  slightly  greater  amount  of  formalin  than  that  used  in  the 
high  temperature  autoclave  and  heated  tube  or  chamber  methods,  the 
results  seem  to  be  equally  as  good.  The  apparatus  is  very  easy  to  use, 
and  is  not  liable  to  get  out  of  order. 

Similar  forms  of  apparatus  are  also  employed,  when  instead  of 
glycerin  the  formalin  is  mixed  with  an  equal  quantity  of  water.  The 
water  is  for  the  purpose  of  giving  additional  moisture  to  the  air,  and, 
at  the  same  time,  like  the  glycerin,  to  prevent  the  change  of  formal- 
dehyde into  inert  substances. 

From  Formalin  in  an  Open  Pan. — A  very  simple  method,  devised 
by  Dr.  R.  J.  Wilson,  is  to  fill  a  tin  pan  with  twelve  ounces  of  formalin 
for  each  1000  cubic  feet  and  put  this  on  an  upright  sheet  of  tin,  which  is 
cut  so  as  to  allow  of  the  entrance  of  air  below  and  yet  protect  the  for- 
malin in  the  pan  from  the  flame.  For  heating  put  under  it  a  small  tin 
can  filled  with  asbestos  packing  which  has  been  soaked  with  wood 
alcohol.  A  still  simpler  method  is  to  hang  sheets  in  a  room  and  throw 
on  them  twelve  ounces  of  formalin  for  each  1000  cubic  feet,  and  leave 
for  ten  hours.  If  the  room  is  tightly  sealed  very  fair  superficial  disin- 
fection will  take  place. 

Lime  Method  of  Generating  Formaldehyde  Gas. — The  use  of  quick- 
lime for  generating  formaldehyde  gas  has  been  practised  by  various 


PRACTICAL  DISIXFECTIOX  AND  STERILIZAT1' >\  127 

observers,  with  varying  results.  The  differing  results  can  probably  be 
explained  by  difference  in  technique  and  in  the  kind  of  lime  used.  It 
JN  absolutely  necessary  to  have  a  quick  slaking  lime  or  a  great  amount 
of  the  gas  will  be  lost  by  polymerization  into  paraformaldehyde  and 
acrose.  Even  with  quick  slaking  lime,  if  it  is  not  spread  in  a  com- 
paratively thin  layer,  polymerization  takes  place;  therefore,  in  applying 
the  method  a  wide  pan  must  be  used.  The  addition  of  concentrated 
sulphuric  acid  to  the  formaldehyde  solution  in  the  proportion  of  10 
per  cent,  immediately  before  using  lessens  the  danger  of  polymeriza- 
tion and  makes  the  evolution  of  the  gas  much  more  rapid.  The  sul- 
phuric acid  must  not  be  added  to  the  formaldehyde  solution  until  just 
before  using,  for  it  causes  rapid  polymerization  in  the  solution.  It 
must  be  remembered,  also,  that  sulphuric  acid  is  a  dangerous  agent, 
and  careless  handling  of  it  might  result  in  serious  burns.  The  technique 
of  the  method  is  as  follows:  To  ten  ounces  of  40  per  cent,  formaldehyde 
solution  slowly  add  one  ounce  of  concentrated  sulphuric  acid;  pour  this 
solution  on  to  two  pounds  of  quicklime  that  has  previously  been  cracked 
into  small  lumps  and  placed  in  a  dairy  pan  not  less  than  twelve  inches 
in  diameter.  The  liberation  of  a  large  amount  of  gas  in  a  short  time 
more  than  compensates  for  the  loss  by  polymerization,  and  disinfection 
is  effected  by  a  quick  union  of  the  gas  and  organisms  to  be  destroyed. 
Saturated  solution  of  aluminum  sulphate  may  be  used  instead  of  con- 
centrated sulphuric  acid,  in  the  proportion  of  one  part  of  aluminum 
sulphate  solution  to  three  parts  of  40  per  cent,  formaldehyde  solution. 
The  mixture  of  aluminum  sulphate  and  formaldehyde  will  stand  for 
considerable  time  without  polymerization.  Good  results  have  been 
obtained  from  pouring  40  per  cent,  solution  of  formaldehyde  into  com- 
mercial permanganate  of  potassium  in  the  proportion  of  six  ounces  of 
permanganate  for  every  pint  of  40  per  cent,  solution  of  formaldehyde. 
Rapid  Generation  of  Formaldehyde  Gas  for  Large  Chambers  by  the  Method 
of  Dr.  R.  J.  Wilson. — The  generator  is  made  of  ordinary  iron  steam  pipe 
and  can  be  manufactured  in  any  pipe-cutting  establishment  in  a  very  few^ 
hours.  It  consists  of  an  outer  steam  jacket  of  six-inch  pipe,  two  feet 
long,  and  capped  at  both  ends.  Through  the  upper  cap  there  is  a  four- 
inch  opening,  with  a  thread,  through  which  projects  an  inner  chamber 
for  formalin.  This  chamber  consists  of  a  four-inch  pipe,  twenty-two 
inches  long,  capped  at  the  upper  end  and  welded  or  capped  at  the 
lower  end.  The  upper  end  of  this  pipe  is  so  threaded  as  to  permit  of 
its  being  screwed  through  the  cap  of  the  steam  jacket  before  that  cap 
is  screwed  on.  The  cap  of  the  formalin  chamber  is  fitted  on  the 
same  thread  that  passes  through  the  cap  of  the  steam  jacket.  The 
in-take  for  steam  is  near  the  top  of  the  steam  jacket,  through  a  half- 
inch  pipe,  and  the  steam  is  controlled  by  a  globe  valve.  The  outlet 
for  steam  or  drip  is  through  a  half-inch  pipe  from  the  bottom  cap  of 
the  chamber  and  is  also  controlled  by  a  globe  valve.  The  in-take  for 
formalin  is  through  the  upper  cap  of  the  formalin  chamber  through  a 
half-inch  pipe  controlled  by  a  globe  valve.  The  outlet  for  formaldehyde 
is  a  half-inch  pipe  through  the  upper  cap  of  the  formalin  chamber. 


128 


PRIXCIPLES  OF  BACTERIOLOGY 


FIG.  66 


This  generator  is  cheap  and  efficient,  but  considerable  care  should 
be  observed  in  operating  it,  as  there  is  a  tendency  to  throw  out  some 

formalin  before  the  gas  begins  to  be  evolved. 
This  is  easily  avoided  by  using  care  in  the 
proper  application  of  the  heat.  These 
generators  have  now  been  in  use  for  three 
years  by  the  New  York  Health  Department, 
and  have  given  complete  satisfaction. 

As  a  result  of  the  investigations  under- 
taken in  the  Department  of  Health  labora- 
tories on  the  use  of  formaldehyde  as  a 
disinfectant,  and  a  consideration  of  the 
work  of  others,  the  conclusions  reached  by 
us  may  be  summarized  as  follows: 

1.  DISINFECTION  OF  INFECTED  DWELL- 
INGS.— Exposed  surfaces  of  walls,  carpets, 
hangings,  etc.,  in  rooms  may  be  super- 
ficially disinfected  by  means  of  formalde- 
hyde gas.  All  apertures  in  the  rooms 
should  be  tightly  closed  and  from  six  to 
twelve  ounces  of  formalin  or  its  equivalent 
used  to  generate  the  gas  for  each  1000 
cubic  feet.  The  time  of  exposure  should 
be  not  less  than  four  hours,  and  a  suitable 
A,  steam  chamber;  B,  formalin  apparatus  should  be  employed.  The  tem- 

chamber:  C,  steam  supply;  D,  d  rip ;  »     ,,  /      i        i  i     i 

E,  inlet  for  formalin;  F,  outlet  tor  perature  of  the  apartment  ^  should  be  as 
formaldehyde.  high  as  possible,  and  certainly  not  below 

50°  F.  With  even  lower  temperature  dis- 
infection is  possible,  but  larger  amounts  of  formalin  must  be  used. 
When  generated  very  rapidly  the  formaldehyde  gives  much  better 
results  than  when  given  off  slowly. 

Under  these  conditions  spore-free  bacteria  and  the  contagion  of  the 
exanthemata  are  surely  destroyed  when  freely  exposed  to  the  action 
of  the  gas.  Spore-bearing  bacteria  are  not  thus  generally  destroyed; 
but  these  latter  are  of  such  rare  occurrence  in  disease  that  in  house 
disinfection  they  may  usually  be  disregarded,  and,  if  present,  special 
measures  can  be  taken. 

The  penetrative  power  of  formaldehyde  gas  in  the  ordinary  room, 
at  the  usual  temperature,  even  when  used  in  double  the  strength  neces- 
sary for  surface  disinfection,  is  extremely  limited,  not  passing,  as  a 
rule,  through  more  than  one  layer  of  cloth  of  medium  thickness.  Arti- 
cles, therefore,  such  as  bedding,  carpets,  upholstery,  clothing,  and  the 
like  should,  when  possible,  be  subjected  to  steam,  hot  air,  or  formal- 
dehyde disinfection  in  special  chambers  constructed  for  the  purpose. 
If  not,  they  must  be  thoroughly  exposed  on  all  sides. 

2.  DISINFECTION  OF  BEDDING,  CARPETS,  UPHOLSTERY,  ETC. — Bed- 
ding, carpets,  clothing,  etc.,  which  would  be  injured  by  steam,  may  be 
disinfected  by  means  of  formaldehyde  gas  in  an  ordinary  steam  disin- 


PRACTICAL  I)ISI\n-;cTI<>\  AM)  STERILIZATION  129 

fecting  chamber,  the  latter  to  be  provided  with  a  heating  and  if  pos- 
sible a  vacuum  apparatus  and  special  apparatus  for  generating  the  gas. 
Where  penetration  through  heavy  articles  is  required  the  gas  should 
be  used  in  the  proportion  of  not  less  than  the  amount  derived  from  thirty 
ounces  of  formalin  for  each  1000  cubic  feet,  the  time  of  exposure  to  be 
not  less  than  eight  hours  and  the  temperature  of  the  chamber  not  below 
110°  F. 

In  order  to  ensure  complete  sterilization  of  the  articles  they  should 
be  so  placed  as  to  allow  of  a  free  circulation  of  the  gas  around  them — 
that  is,  in  the  case  of  bedding,  clothing,  etc.,  these  should  either  be 
spread  out  on  perforated  wire  shelves  or  loosely  suspended  in  the  cham- 
ber. The  aid  of  a  partial  vacuum  facilitates  the  operation.  Upholstered 
furniture  and  articles  requiring  much  space  should  be  placed  in  a  large 
chamber,  or,  better,  in  a  room  which  can  be  heated  to  the  required 
temperature. 

The  most  delicate  fabrics,  furs,  leather,  and  other  articles,  which 
are  injured  by  steam,  hot  air  at  230°  F.,  or  other  disinfectants,  are 
unaffected  by  formaldehyde. 

3.  DISINFECTION   OF   BOOKS. — Books   may   be   satisfactorily   disin- 
fected by  means  of  formaldehyde  gas  in  a  special  room,  or  in  the  ordinary 
steam  chamber,  as  above  described,  and  under  the  same  condition  of 
volume  of  gas,  temperature,  and  time  of  exposure.    The  books  should 
be  arranged  to  stand  as  widely  open  as  possible  upon  perforated  wire 
shelves,  set  about  one  or  one  and  a  half  feet  apart  in  the  chamber.    A 
chamber  having  a  capacity  of  200  to  250  cubic  feet  would  thus  afford 
accommodation  for  about  one  hundred  books  at  a  time. 

Books,  with  the  exception  of  their  surfaces,  cannot  be  satisfactorily 
disinfected  by  formaldehyde  gas  in  the  bookcases  of  houses  and  libraries, 
or  anywhere  except  in  special  chambers  constructed  for  the  purpose, 
because  the  conditions  required  for  their  thorough  disinfection  cannot 
otherwise  be  complied  with. 

The  bindings,  illustrations,  and  print  of  books  are  in  no  way  affected 
by  the  action  of  formaldehyde  gas. 

4.  DISINFECTION  OF  CARRIAGES,  ETC. — Carriages,  ambulances,  cars, 
etc.,  can  easily  be  disinfected  by  having  built  a  small,  tight  building,  in 
which  they  are  enclosed  and  surrounded  with  formaldehyde  gas.     Such 
a  building  is  used  for  disinfecting  ambulances  in  New  York  City.    With 
the  apparatus  there  employed  a  large  amount  of  formalin  is  rapidly 
vaporized,  and  superficial  disinfection  is  completed  in  sixty  minutes. 

5.  ADVANTAGES  OF  FORMALDEHYDE  GAS  OVER  SULPHUR  DIOXIDE 
FOR  DISINFECTION  OF  DWELLINGS. — Formaldehyde  gas  is  superior  to 
sulphur  dioxide  as  a  disinfectant  for  dwellings:  first,  because  it  is  more 
efficient  in  its  action;  second,  because  it  is  less  injurious  in  its  effects 
on  household  goods;  third,  because  when  necessary  it  can  easily  be 
supplied  from  a  generator  placed  outside  of  the  room  and  watched  by 
an  attendant,  thus  avoiding  in  some  cases  danger  of  fire. 

Apart  from  the  cost  of  the  apparatus  and  the  greater  time  involved, 
formaldehyde  gas,  generated  from  commercial  formalin,  is  not  much 

9 


130  PRINCIPLES  OF  BACTERIOLOGY 

more  expensive  than  sulphur  dioxide — viz.,  fifteen  to  twenty  cents  per 
1000  cubic  feet  against  ten  cents  with  sulphur.  Therefore,  we  believe 
that  formaldehyde  gas  is  the  best  disinfectant  at  present  known  for 
the  surface  disinfection  of  infected  dwellings.  For  heavy  goods  it  is 
far  inferior  in  penetrative  power  to  steam;  but  for  the  disinfection  of 
fine  wearing  apparel,  furs,  leather,  upholstery,  books,  and  the  like, 
which  are  injured  by  great  heat,  it  is,  when  properly  employed,  better 
adapted  than  any  other  disinfectant  now  in  use. 

Sulphur  Dioxide  in  House  Disinfection. — Four  pounds  of  sulphur 
should  be  burned  for  every  1000  cubic  feet.  The  sulphur  should  be 
broken  into  small  pieces  and  put  in  a  pan  sufficiently  large  not  to  allow 
the  melted  sulphur  to  overflow.  This  pan  is  placed  in  a  much  larger 
pan  holding  a  little  water.  The  cracks  of  the  room  should  be  carefully 
pasted  up  and  the  door,  after  closing,  also  sealed.  Upon  the  broken 
sulphur  is  poured  three  to  four  ounces  of  alcohol  and  the  whole  lighted 
by  a  match.  The  alcohol  is  not  only  for  the  purpose  of  aiding  the 
sulphur  to  ignite,  but  also  to  add  moisture  to  the  air.  An  exposure 
of  eight  to  twelve  hours  should  be  given. 

Sulphur  fumigation  carried  out  as  above  indicated  is  not  as  efficient 
as  formaldehyde  fumigation,  but  seems  to  suffice  for  surface  disinfec- 
tion for  diphtheria  and  the  exanthemata.  All  heavy  goods  should  be 
removed  for  steam  disinfection  if  there  is  any  possibility  of  the  infection 
having  penetrated  beneath  their  surface.  If  there  is  no  place  for  steam 
disinfection  their  surfaces  should  be  thoroughly  exposed  to  fumigation 
and  then  to  the  air  and  sunlight.  In  many  cases  when  cleanliness  has 
been  observed,  surface  disinfection  of  halls,  bedding,  and  furniture 
may  be  all  that  will  be  required. 

There  is  always  a  very  slight  possibility  of  a  deeper  penetration  of 
infection  than  that  believed  to  have  occurred;  it  is,  therefore,  better 
to  be  more  thorough  than  is  considered  necessary  rather  than  less. 

Sulphur  dioxide  without  the  addition  of  moisture  has,  as  already 
stated  under  the  consideration  of  disinfectants,  very  little  germicidal 
value  upon  dry  bacteria. 

Public  Steam  Disinfecting  Chambers. — These  should  be  of  sufficient 
size  to  receive  all  necessary  goods,  and  may  be  either  cylindrical  or 
rectangular  in  shape,  and  are  provided  with  steam-tight  doors  opening 
at  either  end,  so  that  the  goods  put  in  at  one  door  may  be  removed  at 
the  other.  When  large  the  doors  are  handled  by  convenient  cranes 
and  drawn  tight  by  drop-forged  steel  eye-bolts  swinging  in  and  out  of 
slots  in  the  door  frames.  The  chambers  should  be  able  to  withstand  a 
steam  pressure  of  at  least  one-half  an  atmosphere,  and  should  be  con- 
structed with  an  inside  jacket,  either  in  the  form  of  an  inner  and  outer 
shell  or  of  a  coil  of  pipes.  This  jacket  is  filled  with  steam  during  the 
entire  operation,  and  is  so  used  as  to  bring  the  goods  in  the  disinfecting 
chamber  up  to  the  neighborhood  of  220°  F.  before  allowing  the  steam 
to  pass  in.  ^  This  heats  the  goods,  so  that  the  steam  does  not  condense 
on  coming  in  contact  with  them.  It  is  an  advantage  to  displace  the  air 
in  the  chamber  before  throwing  in  the  steam,  as  hot  air  has  far  less 


r:  \CTICAL  DISIXFECTION  AND  STERILIZATION  131 

germicidal  value  than  steam  of  the  same  temperature.  To  do  this,  a 
vacuum  pump  is  attached  to  the  piping,  whereby  a  vacuum  of  fifteen 
inches  can  be  obtained  in  the  chamber.  The  steam  should  be  thrown 
into  the  chamber  in  large  ajnount,  both  above  and  below  the  goods, 
and  the  excess  should  escape  through  an  opening  in  the  bottom  of  the 
chamber,  so  as  to  more  readily  carry  off  with  it  any  air  still  remaining. 
The  live  steam  in  the  chamber  should  be  under  a  pressure  of  two  to 
three  pounds,  so  as  to  increase  its  action. 

To  disinfect  the  goods,  we  place  them  in  the  chamber,  close  tight  the 
doors,  and  turn  the  steam  into  the  jacket.  After  about  ten  minutes, 
when  the  goods  have  become  heated,  a  vacuum  of  ten  to  fifteen  inches 
is  produced,  and  then  the  live  steam  is  thrown  in  for  twenty  minutes. 
The  steam  is  now  turned  off,  a  vacuum  is  again  formed,  and  the  cham- 
ber again  superheated.  The  goods  are  now  thoroughly  disinfected  and 
dry.  In  order  to  test  the  thoroughness  of  any  disinfection,  or  any  new 
chamber  maximum,  thermometers  are  placed,  some  free  in-  the  cham- 
ber and  others  surrounded  by  the  heaviest  goods.  It  will  be  found 
that,  even  under  a  pressure  of  three  pounds,  live  steam  will  require  ten 
minutes  to  penetrate  heavy  goods. 

The  Disinfection  of  Hands,  Instruments,  Ligatures,  and  Dressings 
for  Surgical  Operations. 

Instruments. — All  instruments,  except  knives,  after  having  been 
thoroughly  cleansed,  are  boiled  for  three  minutes  in  a  1  per  cent,  solu- 
tion of  washing  soda.  Knives,  after  having  been  thoroughly  cleansed, 
are  washed  in  sterile  alcohol  and  wiped  with  sterile  gauze  and  then  put 
into  boiling  soda  solution  for  one  minute.  This  will  not  injure  their 
edges  to  any  great  extent. 

Gauze. — Gauze  is  sterilized  by  moist  heat  either  in  an  Arnold  steam 
sterilizer  for  one  hour  or  in  an  autoclave  for  thirty  minutes.  It  is  placed 
in  a  perforated  cylinder  or  wrapped  in  clean  towels  before  putting  in 
the  sterilizer,  and  only  opened  at  the  operation. 

lodoform  gauze  is  best  made  by  sprinkling  sterile  iodoform  on  plain 
gauze  sterilized  as  described  above. 

Ligatures — Catgut. — Boil  for  one  hour  in  alcohol  under  pressure  at 
about  97°  C.  It  is  often  put  in  sealed  glass  tubes,  which  are  boiled 
under  pressure.  These  remain  indefinitely  sterile.  The  alcohol  does 
not  injure  the  catgut.  If  desired,  the  catgut  can  be  washed  in  ether 
and  can  be  soaked  a  short  time  in  bichloride  before  heating  in  alcohol. 
Boeckman,  of  St.  Paul,  suggested  wrapping  the  separate  strands  of  cat- 
gut in  paraffin  paper  and  then  heating  for  three  hours  at  140°  C.  This 
procedure  prevents  the  drying  out  of  the  moisture  and  fat  from  the 
catgut,  so  that  it  remains  unshri veiled  and  flexible  after  its  exposure. 
Darling,  of  Boston,  tested  this  method  and  found  it  satisfactory.  Dry 
formaldehyde  gas  does  not  penetrate  sufficiently,  and  is  not  reliable. 
Silver  wire,  silk,  silkworm  gut,  rubber  tubing,  and  catheters  are  boiled 
the  same  as  the  instruments. 


132  PRINCIPLES  OF  BACTERIOLOGY 

The  Skin  of  the  Patient. — This  is  washed  thoroughly  with  warm,  green 
soap  solution,  then  with  alcohol,  and  finally  with  1 : 1000  bichloride. 
A  compress  wet  with  a  25  per  cent,  solution  of  green  soap  is  now  placed 
on,  covered  with  rubber  tissue,  and  left  for  three  to  twelve  hours;  and 
after  its  removal  the  skin  is  washed  with  ether,  alcohol,  and  bichloride 
solution,  and  then  covered  with  a  gauze  compress  previously  moistened 
with  a  1 : 1000  bichloride  of  mercury  solution.  At  the  operation  the 
skin  is  again  scrubbed  with  green  soap  solution  followed  by  ether, 
alcohol,  and  then  with  the  bichloride  of  mercury  solution.  In  some 
places  the  bichloride  compress  is  replaced  one  hour  before  the  opera- 
tion by  a  pad  wet  in  10  per  cent,  solution  of  formalin. 

The  Hands. — Fiirbinger's  method,  slightly  modified,  is  now  much 
used,  and  gives  good  results.  The  hands  are  washed  in  hot  soap  and 
water  for  five  minutes,  using  the  nail  brush.  They  are  then  soaked  in 
alcohol  for  one  minute  and  scrubbed  with  a  sterile  brush.  They  are 
finally  soaked  in  a  1 : 1000  bichloride  of  mercury  solution  for  three 
minutes.  Another  method  which  gives  good  results  is  as  follows :  Skin 
of  operator  is  scrubbed  for  five  minutes  with  green  soap  and  brush, 
then  washed  in  chloride  of  lime  and  carbonate  of  soda  in  proportions 
to  make  a  good  lather;  washed  off  in  sterile  water,  and  then  scrubbed 
with  brush  in  warm  bichloride  solution  1 : 1000. 

Sterilized  rubber  gloves  are  now  being  used  more  and  more  in  opera- 
tions. The  gloves  can  be  sterilized  by  being  left  for  one  minute  in  boil- 
ing 1  per  cent,  soda  solution,  or  they  can  be  sterilized  by  steam. 

The  surgeon's  gowns  and  caps  are  sterilized  by  steam.  Mucous 
membranes,  as  those  of  the  mouth  and  throat,  are  cleansed  by  a  solu- 
tion consisting  of  equal  parts  of  peroxide  of  hydrogen  and  lime-water. 
In  the  nostrils  it  is  better  to  employ  the  milder  solutions,  such  as  diluted 
Dobell's  or  Listerine.  These  are  also  used  in  the  mouth  instead  of  the 
peroxide. 

The  vagina  is  swabbed  out  thoroughly  with  sterile  warm  soap  and 
water,  and  then  irrigated  with  a  2  per  cent,  carbolic  acid  or  a  1 : 1000 
bichloride  of  mercury  solution. 

Hypodermic  syringes  and  other  syringes  when  not  boiled  are  steril- 
ized by  drawing  up  into  them  boiling  water  a  number  of  times  and  then 
finally  a  5  per  cent,  solution  of  carbolic  acid,  the  acid  after  three  minutes 
to  be  washed  out  by  boiling  water.  If  cold  water  is  used  the  carbolic 
solution  should  remain  in  the  barrel  for  ten  minutes.  Great  care  should 
be  taken  to  wash  out  all  possible  organic  matter  before  using  the  car- 
bolic acid  or  boiling  to  sterilize.  Syringes  made  entirely  of  glass  or  of 
glass  and  asbestos  can  be  boiled  in  soda  solution. 

The  Sterilization  of  Milk. 

Bacteria  when  allowed  to  develop  in  milk  produce  fermentation 
(souring)  and  render  the  milk  unfit  to  be  used  as  an  article  of  food, 
especially  for  infants.  Milk  as  it  reaches  the  city  contains  enormous 
numbers  of  germs,  and  these  will  produce  fermentation,  even  though 


PRACTICAL  7>/>7\ /••/•:<  77" .Y    \\I>  > Tl:, .'//.//.  1  TION  133 

the  milk  be  kept  on  ice.  Unclean  vessels  hasten  this  process.  No  matter 
how  good  milk  may  be  in  the  morning,  when  comparatively  fresh, 
toward  evening,  unless  it  has  been  partly  or  completely  sterilized,  it 
may  be  dangerous  to  an  infant,  and  may,  especially  in  summer,  cause 
fatal  illness,  even  though  it  still  tastes  sweet. 

Complete  sterilization  destroys  all  the  germs  in  milk,  and  so  prevents 
permanently  fermentative  changes.  By  partial  sterilization  most  of 
the  germs  which  are  not  in  the  spore  form  may  be  destroyed,  so  that 
the  milk  will  remain  wholesome  for  at  least  twenty-four  hours  in  the 
warmest  weather. 

Milk  is  best  sterilized  by  heat,  for  nearly  all  chemicals,  such  as 
boric  acid,  salicylic  acid,  and  formalin,  are  not  only  slightly  deleterious 
themselves  but  also  make  the  milk  less  digestible,  and,  therefore,  less 
fit  for  food.  It  may  be  sterilized  at  a  high  or  low  temperature — that 
is,  at  the  boiling  temperature — or  at  a  lower  degree  of  heat,  obtained 
by  modifying  the  steaming  process. 

It  has  been  found  that  milk  sterilized  at  a  high  temperature  (100°  C.) 
is  not  desirable  for  prolonged  use,  as  the  high  temperature  causes  cer- 
tain changes  in  the  milk  which  make  it  less  suitable  as  a  food  for  infants. 
These  changes  are  almost  altogether  avoided  if  a  temperature  below 
80°  C.  is  used.  It  is  recommended,  therefore,  that  the  lowest  tempera- 
ture be  used  for  partial  sterilization  which  will  keep  the  milk  whole- 
some for  twenty-four  hours  in  the  warmest  weather  and  kill  the  tuber- 
cle, typhoid,  and  other  non-spore-bearing  bacilli.  Raising  the  milk 
to  a  temperature  of  70°  C.  for  fifteen  minutes  or  80°  C.  for  twelve 
minutes  will  accomplish  this.  One  of  the  many  forms  of  apparatus  is 
the  following: 

(a)  A  tin  pail  or  pot,  about  ten  inches  deep  by  nine  inches  in  diame- 
ter, provided  with  the  ordinary  tin  cover  which  has  been  perforated 
with  eight  holes  each  an  inch  in  diameter. 

(6)  A  wire  basket,  with  eight  nursing  bottles  (as  sold  for  this 
purpose  in  the  shops). 

(c)  Rubber  corks  for  the  bottles  and  a  bristle  brush  for  cleaning 
them. 

Directions  (Koplik). — Place  the  milk,  pure  or  diluted  (as  the 
physician  may  direct),  in  the  nursing  bottles  and  place  the  latter 
in  the  wire  basket.  Put  only  sufficient  milk  for  one  nursing  in  each 
bottle.  Do  not  cork  the  bottles  at  first. 

Having  previously  poured  about  two  inches  of  water  in  the  tin  pail 
or  pot  and  brought  it  to  the  boiling  point,  lower  the  basket  of  nursing 
bottles  slowly  into  the  pot.  Do  not  allow  the  bottles  to  touch  the  water 
or  they  will  crack.  Put  on  the  perforated  cover  and  let  the  steaming 
continue  for  ten  minutes;  then  remove  the  cover  and  firmly  cork  each 
bottle.  After  replacing  the  cover,  allow  the  steaming  to  continue  for 
fifteen  minutes.  The  steam  must  be  allowed  to  escape  freely  or  the 
temperature  will  rise  too  high. 

The  process  is  now  completed.  Place  the  basket  of  bottles  in  a 
cool,  dark  place  or  in  an  ice-chest.  The  bottles  must  not  be  opened 


134  PRINCIPLES  OF  BACTERIOLOGY 

until  just  before  the  milk  is  to  be  used,  and  then  it  may  be  warmed  by 
plunging  the  bottle  in  warm  water.  If  properly  prepared  the  milk 
will  taste  but  little  like  boiled  milk. 

The  temperature  attained  under  the  conditions  stated  above  will 
not  exceed  in  extreme  cases  87°  C.  (188°  F.). 

Milk  should  be  sterilized  when  it  is  as  fresh  as  possible,  and  only 
sufficient  milk  for  twenty-four  hours  should  be  sterilized  at  one  time. 
If  after  nursing  the  infant  leaves  some  milk  in  the  bottle  this  should 
be  thrown  away. 

Care  of  the  Bottles. — After  nursing,  the  bottles  should  be  filled  with 
a  strong  solution  of  washing  soda,  allowed  to  stand  twenty-four  hours, 
and  then  carefully  cleaned  with  a  bristle  (bottle)  brush.  The  rubber 
corks  and  nipples  should  be  boiled  after  using  in  strong  soda  solution 
for  fifteen  minutes  and  then  rinsed  and  dried. 

After  sterilizing  rnilk  should  never  be  put  into  unsterilized]bottles, 
as  this  will  spoil  it. 

A  different  but  admirable  method  is  the  one  devised  by  Dr.  Free- 
man.1 Here  a  pail  is  filled  to  a  certain  mark  with  water,  and  then  placed 
on  the  stove  until  the  water  boils.  It  is  then  removed,  and  immediately 
a  milk-holder,  consisting  of  a  series  of  zinc  cylinders,  is  lowered  with 
its  milk  bottles  partially  full  of  milk.  The  cover  is  again  applied. 
The  heat  of  the  outside  water  raises  the  temperature  of  the  milk  in 
ten  minutes  to  75°  C.  (167°  F.),  and  holds  it  nearly  at  that  point  for 
some  time.2  After  twenty  minutes  the  milk  is  removed,  placed  in  cold 
water,  and  quickly  cooled.  The  milk  is  kept  in  the  ice-chest  until 
used. 

1  Agent  for  Pasteurizer,  James  Dougherty,  411  W.  59th  St. 

2  A  temperature  of  75°  C.  is  advised  in  Pasteurizing  milk,  instead  of  65°  C.,  which  would  ordi- 
narily suffice  to  kill  all  bacteria  free  of  spores,  because  of  the  fact  pointed  out  by  Theobald  Smith, 
that  the  bacteria  embedded  in  the  pellicle  which  forms  on  the  surface  are  more  resistant  than  those 
surrounded  by  fluid. 


CHAPTER    XL 

THE  USE  OF  ANIMALS  FOR  DIAGNOSTIC  AND  TEST  PURPOSES. 

SUITABLE  animals  are  necessarily  employed  for  many  bacteriological 
purposes.  1.  To  obtain  a  development:  Thus  they  may  be  used  as  a 
soil  for  bacterial  growth,  when,  as  in  the  case  of  tubercle  bacilli,  we 
cannot  get  a  growth  in  the  dead  culture  media.  For  this  reason  material 
suspected  to  contain  tubercle  bacilli  is  injected  into  rabbits  or  guinea- 
pigs,  with  the  knowledge  that,  if  present,  although  in  too  small  numbers 
to  be  detected  by  microscopic  or  culture  methods,  they  will  develop 
in  the  animals'  bodies,  and  thus  reveal  themselves.  The  same  may  be 
true  of  glanders,  tetanus,  and  anthrax  bacilli,  of  pneumococci,  of  other 
bacteria,  and  of  protozoa.  2.  To  cause  an  increase  of  one  variety  of 
organisms  in  a  mixture:  An  injection  of  sputum  subcutaneously  in 
rabbits  may  give  rise  to  a  pure  pneumococctis  septicaemia  or  a  pure 
tuberculosis.  3.  To  test  virulence:  Animals  are  used  to  test  the  viru- 
lence or  toxin  production  of  organisms,  where,  as  in  the  case  of  diph- 
theria, we  have  very  virulent,  attenuated,  and  non-virulent  bacilli  of, 
so  far  as  we  know,  identical  cultural  characteristics.  Here  the  injec- 
tion of  a  susceptible  animal,  such  as  the  guinea-pig,  is  the  only  way 
that  we  can  differentiate  between  those  capable  of  producing  diseases 
from  those  that  are  harmless.  Still  another  use  of  animals  is  to  dif- 
ferentiate between  two  virulent  organisms,  which,  though  entirely 
different  in  their  specific  disease  poisons,  are  yet  so  closely  allied  mor- 
phologically and  in  culture  characteristics  that  they  cannot  always  be 
separated  except  by  studying  their  action  in  the  animal  body  both 
without  and  under  the  influence  of  specific  serums  upon  them.  In 
this  way  the  typhoid  and  colon  bacilli  may  be  separated,  or  the  pneu- 
mococcus  and  streptococcus.  4.  To  test  the  antitoxic  or  bactericidal 
strength  of  sera:  Diphtheria  antitoxin  in  different  amounts  is  added 
to  one  hundred  fatal  doses  of  diphtheria  toxin  and  injected  into  guinea- 
pigs,  and  streptococcus  immunizing  serum  is  mixed  with  living  strepto- 
cocci and  injected  into  the  vein  of  a  rabbit.  5.  To  produce  antitoxic, 
bactericidal,  or  agglutinating  sera. 

The  Inoculation  of  Animals. — The  inoculation  of  animals  may  be 
made  either  through  natural  channels  or  through  artificial  ones: 

1.  Cutaneous.     Cultures  are  rubbed  into  the  abraded  skin. 

2.  Subcutaneous.     The  bacteria  are  injected  by  means  of  a  hypo- 
dermic needle  under  the  skin,  or  are  introduced  by  a  platinum  loop 
into  a  pocket  made  by  an  incision. 

3.  Intravenous.      The  bacteria  are  injected    by  means  of  a  hypo- 
dermic needle  into  the  vein.    This  is  usually  carried  out  in  the  ear  vein 


PRINCIPLES  OF  BACTERIOLOGY 

of  the  rabbit.  If  rabbits  are  placed  in  a  holder,  so  that  the  rabbit 
remains  quiet  and  only  the  head  projects,  it  is  usually  easy  to  pass  a 
small  needle  directly  into  one  of  the  ear  veins,  especially  those  running 
along  their  edges.  If  the  ear  is  first  moistened  with  a  3  per  cent,  car- 
bolic acid  solution,  and  then  supported  between  the  finger  inside  and 
the  thumb  outside,  the  vein  is  usually  clearly  seen  and  entered  with 
ease,  if  a  small,  sharp  needle  is  held  almost  parallel  with  the  ear  surface 
and  gently  pushed  into  it.  When  no  holder  is  present,  the  rabbit  can 
be  held  by  an  assistant  seizing  the  forelegs  in  one  hand  and  the  hind 
in  another  and  holding  the  rabbit  head  downward. 

4.  Into  the  anterior  chamber  of  the  eye. 

5.  Into  the  body  cavities.    The  peritoneal  and  less  often  the  pleural 
cavities  are  used  for  bacterial  injection.     The  hypodermic  needle  is 
usually  employed,  less  often  a  glass  tube  drawn  out  to  a  fine  point. 
The  needle  or  the  pointed  glass   tube  is  gently  pushed  through  the 
abdominal  wall,  moved  about  to  ensure  its  freedom  from  the  intestines, 
and  the  fluid  injected. 

6.  By  inhalation.    This  method  is  carried  out  by  forcing  the  animal 
to  inhale  an  infected  spray  or  dust. 

7.  By  the  trachea.    This  method  is  carried  out  by  making  an  incision 
in  the  trachea  and  then  inoculating  the  mucous  membrane  or  injected 
substances  into  the  trachea  and  bronchi. 

8.  Through  the  intestinal  tract  by  swallowing  or  the  passage  of  a 
rubber  tube. 

9.  Into  the  brain  subtance  or  ventricles  after  trephining. 

In  these  injections  guinea-pigs  are  held,  as  a  rule,  by  an  assistant 
grasping  in  one  hand  the  forelegs  and  in  the  other  the  hindlegs. 

Rabbits  can  be  held  in  the  same  manner,  or,  better,  placed  in  some 
holder  or  strung  up  by  their  hindlegs. 

Mice,  which  are  usually  inoculated  subcutaneously  in  the  body  or 
at  the  root  of  the  tail,  are  best  placed  in  a  mouse  holder,  but  can  be 
inoculated  by  grasping  the  tail  in  a  pair  of  forceps,  and  then,  while 
allowing  the  mouse  to  hang  head  downward  in  a  jar,  a  glass  plate  is 
pushed  across  the  top  until  only  space  for  its  tail  is  left. 

All  these  methods  must  be  carried  out  with  the  greatest  care  as  to 
cleanliness,  the  hair  being  clipped  and  the  skin  partially,  at  least,  dis- 
infected. The  operator  must  be  careful  not  to  infect  himself  or  his 
surroundings.  After  the  inoculations  the  animals  should  be  given  the 
best  of  care,  unless,  for  special  purposes,  we  want  to  study  them  under 
unusual  conditions.  For  food,  rabbits  and  guinea-pigs  require  only 
carrots  and  hay. 

^  If  animals  die,  autopsy  should  be  made  at  the  earliest  moment  pos- 
sible, for  soon  after  death  some  of  the  species  of  the  bacteria  in  the 
intestines  are  able  to  penetrate  through  the  intestinal  walls  and  infect 
the  body  tissues.  If  delay  is  unavoidable,  the  animals  should  be  placed 
immediately  in  a  cold  place.  In  making  cultures  from  the  dead  bodies 
the  greatest  care  should  be  taken  to  avoid  contamination.  The  skin 
should  be  disinfected,  and  any  dust  prevented  by  wetting  with  a  5  per 


USE  OF  AMMALS  FOR  DIAGNOSTIC  AXD  TEST  PURPOSES     137 

cent,  solution  of  carbolic  acid.  All  instruments  are  sterilized  by  I ><  til- 
ing in  3  per  cent,  soda  solution  for  five  minutes.  Changes  of  knives, 
scissors,  and  forceps  should  be  made  as  frequently  as  the  old  ones 
become  infected.  When  organs  are  examined  the  portion  of  the  sur- 
face through  which  an  incision  is  to  be  made  must  be  sterilized,  if 
there  is  danger  that  the  surrounding  cavity  is  infected,  by  searing  with 
the  flat  blade  of  an  iron  spatula  which  has  been  heated  to  a  dull-red 
heat.  Tissues  if  removed  should  be  immediately  placed  under  cover 
after  removal  so  as  not  to  become  infected.  Sterile  deep  Petri  plates 
are  useful  for  this  purpose. 

When  it  is  necessary  to  transport  tissues  some  distance  they  should 
be  wrapped  in  bichloride  cloths  and  sent  to  the  point  of  destination 
as  soon  as  possible.  In  warm  weather  they  may  be  kept  cool  by  sur- 
rounding the  vessel  which  contains  them  with  ice. 

Animals  rarely  show  the  same  gross  lesions  as  man  when  both  suffer 
from  the  same  infection.  The  cell  changes  are  similar,  and,  also,  so  far 
as  we  can  test  them,  the  curative  or  immunizing  effects  of  protective 
serums. 

Leukocytes  for  Testing  Phagocytosis. — Inoculate  into  the  pleural  cavity 
of  a  rabbit  5  c.c.  of  a  thick  suspension  of  aleuronat  powder  in  a  boiled 
starch  solution.  The  solution  should  be  thick  enough  to  hold  the 
aleuronat  in  suspension.  A  20  to  25  per  cent,  solution  of  peptone 
gives  good  results.  The  fluid  is  withdrawn  eighteen  to  twenty-four 
hours  after  the  injection. 


CHAPTER    XII. 

THE  PROCURING  OF  MATERIAL  FOR  BACTERIOLOGICAL  EXAMI- 
NATION FROM  THOSE  SUFFERING  FROM  DISEASE. 

A  LONG  experience  has  taught  me  that  physicians  very  frequently 
take  a  great  amount  of  trouble,  and  yet,  on  account  of  not  carrying  out 
certain  simple  but  necessary  precautions,  make  worthless  cultures  or 
send  material  almost  useless  for  bacteriological  study. 

In  making  cultures  from  diseased  tissues  various  procedures  may 
be  carried  out,  according  to  the  facilities  which  the  physician  has  and 
the  kind  of  information  that  he  desires  to  obtain.  From  the  dead  body 
culture  material  should  be  removed  at  the  first  moment  possible  after 
death.  Every  hour's  delay  makes  the  results  less  reliable.  From  both 
dead  and  living  tissues  the  less  the  alteration  that  occurs  in  any  sub- 
stance between  its  removal  from  the  body  and  its  inoculation  upon  or 
in  culture  media  or  animals  the  more  exact  the  information  which  will 
be  obtained  from  its  examination.  If  the  material  is  allowed  to  dry 
many  bacteria  will  be  destroyed  in  the  process,  and  certain  forms  which 
were  present  will  be  obliterated  or,  at  least,  entirely  altered  in  the  pro- 
portion which  they  bear  to  others.  If  possible,  therefore,  culture  media 
should  be  inoculated  in  the  neighborhood  of  the  patient  or  dead  body. 
For  that  purpose  a  bacteriologist  should  take  the  most  suitable  of  the 
culture  media  to  the  bedside  or  autopsy  table.  Such  a  list  of  media, 
if  fairly  complete,  would  comprise  nutrient  bouillon  alone  and  mixed 
with  one-third  its  quantity  of  ascitic  fluid,  slanted  nutrient  agar,  slanted 
agar  streaked  with  rabbit  or  human  blood,  and  firmly  solidified  slanted 
blood  serum.  If  only  one  variety  of  media  is  to  be  used  the  solidified 
blood  serum  is  most  useful  for  parasitic  bacteria,  and  this  can  be  easily 
carried  by  the  physician  and  inoculated  by  him,  even  if  he  is  not 
very  familiar  with  bacteriological  technique.  The  material  must  be 
obtained  in  different  ways,  according  to  the  nature  of  the  infection. 

For  the  detection  of  the  bacteria  causing  septicaemia  we  are  met 
with  the  difficulty  that  there  are  apt  to  be  very  few  organisms  present 
in  the  blood  until  shortly  before  death.  It  will,  therefore,  be  use- 
less to  take  only  a  drop  of  blood  for  cultures,  as  even  when  present 
there  may  not  be  more  than  eight  or  ten  organisms  in  a  cubic  centi- 
metre. If  cultures  are  to  be  made  at  all,  it  is,  therefore,  best  to  make 
them  correctly  by  taking  from  5  to  20  c.c.  of  blood  by  means  of  a 
sterile  hypodermic  needle,  or  a  suitable  glass  tube  armed  with  a  hypo- 
dermic needle,  from  the  vein  of  the  arm,  after  proper  cleansing  of  the 
skin  and  a  tiny  incision.  Into  each  of  five  different  tubes  containing 
bouillon  we  add  1  c.c.  of  blood,  and  into  a  flask  containing  100  c.c.  we 


<>!•    MATERIAL  FOR  I-XAM I \ ATION  139 

add  5  c.c.  We  have  made  by^this  mixture  of  blood  and  bouillon  a 
most  suitable  medium  for  the  growth  of  all  bacteria  which  produce 
septicaemia,  and  at  the  same  time  have  added  a  sufficient  quantity  of 
blood  to  ensure  us  the  best  possible  chance  of  having  added  some  of 
the  bacteria  producing  the  disease.  We  also  add  to  each  several 
tubes  of  melted  nutrient  agar,  at  40°  C.,  1  c.c.  of  blood  and  pour  into 
Petri  plates,  so  as  to  indicate  roughly  the  number  of  organisms  present 
if  they  happen  to  be  in  abundance.  From  wounds,  abscesses,  cellulitis, 
etc.,  the  substance  for  bacteriological  examination  can,  as  a  rule,  best  be 
obtained  by  means  of  a  syringe,  or  when  opened ,  by  small  rods  armed 
with  a  little  absorbent  cotton.  A  number  of  these  can  be  carried  in  a 
test-tube.  Both  rods  and  tubes  must  be  sterile.  The  swab  is  inserted 
in  (the  wound,  then  streaked  gently  over  the  oblique  surface  of  the 
nutrient  agar  in  one  tube,  over  the  blood  serum  in  another,  and  then 
inserted  in  the  bouillon.  Finally,  either  at  the  bedside  or  in  the  labo- 
ratory, material  is  thinly  streaked  over  the  surface  of  nutrient  agar 
contained  in  several  Petri  dishes.  We  inoculate  several  varieties  of 
media,  with  the  hope  that  one  at  least  will  prove  a  suitable  soil  for  the 
growth  of  the  organisms  present.  From  surface  infections  of  mucous 
membranes,  as  in  the  nose,  throat,  vagina,  etc.,  the  swab,  again,  is 
probably  the  most  useful  instrument  for  obtaining  the  material  for 
examination.  The  greatest  care,  of  course,  must  be  used  in  all  cases 
to  remove  the  material  for  study  without  contaminating  it  in  any  way 
by  other  material  which  does  not  belong  to  it.  Thus,  for  instance, 
if  we  wish  to  obtain  material  from  an  abscess  of  the  liver,  where  the 
organ  lies  in  a  peritoneal  cavity  infected  with  bacteria,  here  one  must 
first  absolutely  sterilize  the  surface  of  the  liver  by  pressing  on  it  the 
blade  of  a  hot  iron  spatula  before  cutting  into  the  abscess,  so  that  we 
may  not  attribute  the  infection  which  caused  the  abscess  to  the  germs 
which  we  obtained  from  the  infected  surface  of  the  liver.  From  such 
an  organ  as  the  uterus  it  is  only  with  the  greatest  care  that  we  can  avoid 
outside  contamination,  and  only  an  expert  bacteriologist  familiar  with 
such  material  will  be  able  to  eliminate  the  vaginal  from  the  uterine 
bacteria. 

A  statement  of  the  conditions  under  which  materials  are  obtained 
should  always  accompany  them  when  sent  to  the  laboratory  for  exami- 
nation, even  if  the  examination  is  to  be  made  by  the  one  who  made 
the  cultures.  These  facts  should  be  noted,  or  otherwise  at  some  future 
date  they  may  be  forgotten  and  misleading  information  sent  out.  The 
work  of  obtaining  material  for  examination  without  contamination 
is  at  times  one  of  extreme  difficulty.  It  simply  must  be  remembered 
that  if  contamination  does  take  place  our  results  may  become  entirely 
vitiated,  and  if  the  difficulties  are  so  great  that  we  cannot  avoid  it,  it 
may  simply  mean  that  under  such  conditions  no  suitable  examination 
can  be  made.  Where  the  substance  to  be  studied  cannot  be  immedi- 
ately subjected  to  cultures  or  animal  inoculations  it  should  be  trans- 
ferred in  a  sterile  bottle  as  soon  as  possible  to  a  location  where  the 
cultures  can  be  made.  If  for  any  reason  delay  must  take  place,  the 


140  PRINCIPLES  OF  BACTERIOLOGY 

material  should  at  least  be  put  in  a  refrigerator,  where  cold  will  both 
prevent  any  further  growth  of  some  varieties  of  bacteria  and  lessen 
the  danger  of  the  death  of  others.  After  having  made  the  cultures, 
some  of  the  infected  material  should  always  be  smeared  on  a  couple  of 
clean  slides  or  cover-glasses  and  allowed  to  dry.  These  can  be  stained 
and  examined  later,  and  may  give  much  valuable  information. 

In  obtaining  samples  of  fluid,  such  as  urine,  feces,  etc.,  the  bottles 
in  which  they  are  placed  should  always  be  sterile,  and,  of  course,  no 
antiseptic  should  be  added.  It  is  necessary  to  clearly  explain  this  to  the 
nurse,  for  she  has  probably  been  instructed  to  add  disinfectants  to  all 
discharges.  Disinfected  material  is,  of  course,  entirely  useless  for 
bacteriological  investigations.  It  cannot  be  too  much  emphasized 
that  materials  which  are  not  immediately  used  should  be  sent  to  the 
laboratory  as  quickly  as  possible,  for  in  such  substances  as  feces,  where 
enormous  numbers  of  various  kinds  of  bacteria  are  present,  those 
which  we  seek  most,  such  as  the  typhoid  bacilli,  frequently  succumb 
to  the  deleterious  products  of  the  other  bacteria  present.  Even  when 
abundantly  present  living  typhoid  bacilli  may  entirely  disappear  from 
the  feces  in  the  course  of  even  twelve  hours,  while  at  other  times  they 
may  remain  present  for  weeks.  These  differences  depend  on  the  asso- 
ciated organisms  present,  the  chemical  constitution  of  the  feces  or 
urine,  and  the  conditions  under  which  the  material  is  obtained. 

Not  only  for  obtaining  fluid  for  agglutination  purposes,  but  also  for 
examination  for  peculiar  bodies  in  the  exanthemata,  blister  fluid  is 
valuable.  A  blister  can  be  raised  quickly  by  placing  a  little  strong 
ammonia  on  the  skin  and  covering  with  a  watch-glass,  or  more  slowly 
by  a  caritharides  plaster. 


CHAPTER   XIII. 

THE  RELATION  OF  BACTERIA  TO  DISEASE. 

Ix  preceding  chapters  we  have  considered  the  chemical  effects  of 
bacteria  and  their  ferments  on  dead  organic  substances.  Now  we  have 
to  consider  the  growth  of  bacteria  in  the  living  host  and  the  results  of 
such  development.  While  it  is  true  that  there  is  a  great  difference 
between  living  and  dead  matter,  and  that,  therefore,  the  living  animal 
cannot  be  looked  upon  as  merely  a  quantity  of  peculiarly  specialized 
material  to  be  used  for  food  for  bacterial  growth,  still,  in  a  very 
real  sense,  we  are  warranted  in  considering  the  infected  living  body 
as  a  food  mass  subject  to  bacterial  growth.  The  difference  is  that 
besides  the  chemical  substances,  temperature,  and  conditions  inherent 
to  the  fluids  of  the  living  body  and  its  tissues,  micro-organisms  have 
also  to  reckon  with  the  constant  production  of  new  substances  by  the 
living  cells  of  the  invaded  organism,  which  maybe  antagonistic  to  them. 
In  the  production  of  lesions  by  micro-organisms  there  are  four  main 
factors  involved — viz.,  on  the  part  of  micro-organisms,  the  power  to 
elaborate  poison  and  the  ability  to  multiply;  on  the  part  of  the  body, 
the  degree  of  sensitiveness  to  the  poisons  of  the  bacteria  and  the  tendency 
to  produce  antitoxic  or  bactericidal  substances.  Xo  known  variety  of 
bacterial  cell  has  as  a  single  organism  the  ability  to  produce  enough 
poison  to  do  appreciable  injury  in  the  body,  nor  is  it  probable  that  there 
is  any  variety  which,  if  it  multiplied  in  the  body  to  the  extent  that  some 
pathogenic  bacteria  are  capable  of,  would  not  produce  disease.  As 
already  mentioned,  varieties  of  bacteria  even  under  similar  conditions 
differ  enormously  in  the  amount  of  poison  which  they  produce  and  in 
their  ability  after  gaining  entrance  to  multiply  in  the  body. 

To  understand  the  bacterial  factor  in  the  production  o*f  disease  we 
must  recognize  that  both  the  body  invaded  and  the  bacteria  which 
invade  are  living  organisms,  arid  that  the  products  of  the  cellular  activity 
of  the  body  act  on  the  bacteria  at  the  same  time  the  bacterial  products 
act  upon  the  human  body.  Just  as  there  are  different  races  and  species 
of  animals  having  dissimilar  characteristics,  there  are  different  races 
and  species  among  bacteria,  and  just  as  the  descendants  of  one  animal 
species  under  changing  conditions  gradually  become  diverse,  so  do  the 
descendants  of  one  bacterial  species.  In  fact,  the  rapidity  of  the  devel- 
opment of  new  generations  of  bacteria  allow  in  them  of  much  quicker 
changes  under  new  conditions  than  are  possible  in  the  higher  animals 
and  plants.  Considering  these  and  other  facts,  we  can  readily  under- 
stand how  the  different  types  of  bacteria  do  not  grow  equally  well  in 
every  variety  of  animal,  and  after  discovering  that  there  are  variations 


PRIXCIPLES  OF  BACTERIOLOGY 

in  the  bactericidal  properties  of  the  blood  from  day  to  day  we  are  not 
surprised  that  they  do  not  find  the  body  of  the  same  animal  always 
equally  suitable.  The  study  of  bacteria  in  the  more  simple  and  known 
conditions  of  artificial  culture  media  has  shown  us  how  extremely 
sensitive  many  bacteria  are  to  slight  chemical,  and  other  changes.  In 
media  conditions  favorable  to  growth  may  still  be  unfavorable  for  toxin 
production. 

If  we  take  specimens  of  diphtheria  bacilli  from  three  different  cases 
of  diphtheria,  we  sometimes  find  that  on  growing  them  for  several  days 
in  suitable  bouillon  one  will  have  produced  poison  in  the  culture  fluid 
to  such  a  degree  that  a  single  drop  suffices  to  kill  a  large  guinea-pig; 
the  second,  grown  in  a  similar  manner,  will  kill  another  animal  of  the 
same  size  with  half  a  drop;  while  the  third  will  kill  with  one-tenth 
of  a  drop.  This  illustrates  the  important  fact  that  different  varieties 
of  the  same  bacillus  have  different  toxin-producing  powers  under  the 
same  conditions — that  is,  that  the  conditions  that  are  suitable  for  the 
full  development  of  the  functions  of  one  strain  are  not  so  for  another 
strain. 

Let  us  now  cultivate  these  same  strains  in  bouillon  which  is  a  little 
too  acid  or  a  little  too  alkaline  for  their  maximum  development,  and 
we  shall  find  that  while  all  of  them  will  grow,  only  one  and  probably 
that  one  which  produced  the  most  toxin  under  favorable  conditions  will 
continue  to  develop  it,  while  the  others  will  fail  to  produce  any  specific 
poison.  This  illustration  makes  clear  one  reason  for  the  variation  in 
severity  among  different  cases  in  an  epidemic,  since  the  conditions  in 
one  throat  may  favor  growth  but  not  toxin  production,  while  in  another 
throat  both  are  favored.  The  fact  that  growth  of  bacteria  may  occur  in 
the  body  and  yet  no  specific  poison  be  produced,  and  that,  of  the  same 
species  of  bacteria,  some  varieties  are  capable  of  producing  toxin  under 
less  favorable  circumstances  than  others,  is  very  important  to  remember. 

The  cultivation  of  the  tetanus  bacillus  also  furnishes  some  interesting 
facts  which  illustrate  the  complicated  ways  in  which  the  growth  of 
varieties  of  bacteria  are  hindered  or  assisted.  The  tetanus  bacillus, 
when  placed  in  suitable  media,  will  not  grow  except  in  the  absence  of 
oxygen;  but  place  it  under  the  same  conditions,  together  with  a  micro- 
organism which  actively  assimilates  oxygen,  and  the  two  in  association 
will  grow  in  the  presence  of  air.  As  a  rule,  when  tetanus  bacilli  are 
driven  into  the  flesh  by  a  dirty  nail  or  blank  cartridge  plug/ aerobic 
bacteria  are  driven  in  also  and  so  help  to  further  infection. 

The  influenza  bacillus  is  a  striking  example  of  the  special  require- 
ments of  certain  bacteria.  On  culture  media  it  will  thrive  only  in  the 
presence  of  haemoglobin. 

It  is  evident,  therefore,  that  for  each  variety  of  organism  there  are 
special  conditions  requisite  for  growth,  and  that  a  temperature,  degree 
of  acidity,  kind  of  food,  supply  of  oxygen,  etc.,  suitable  for  one  may  be 
utterly  unsuitable  for  another;  that, 'still  further,  when  two  organisms 
grow  together  one  may  so  alter  some  of  these  conditions  as  to  render 
unsuitable  ones  suitable,  and  vice  versa. 


THE  RELATION  OF  BACTERIA  TO  DISEASE  143 

Let  us  now  consider  some  of  the  facts  which  have  been  observed 
concerning  the  growth  of  bacteria  in  the  living  body  as  contrasted  with 
culture  media.  In  the  first  place,  it  has  been  learned,  as  will  be  described 
in  the  latter  part  of  the  book,  that  each  variety  of  bacteria  can  incite 
only  certain  types  of  infection.  Indeed,  because  of  this  fact,  the  majority 
of  bacteria  which  excite  disease  can  be  traced  back  for  thousands  of 
years  by  means  of  the  records,  these  parasitic  bacteria  breeding  true 
and  keeping  distinct  from  the  great  mass  of  bacteria  occurring  in  the 
air,  water,  and  soil. 

Parasitic  bacteria  have  gradually  adapted  themselves  not  only  to 
certain  species  of  animals,  but  to  certain  circumscribed  areas  of  the 
body.  Thus  the  diphtheria  bacilli  grow  chiefly  upon  the  mucous  mem- 
branes of  the  respiratory  tract,  but  cannot  develop  in  the  blood  or  in 
the  subcutaneous  tissues.  The  cholera  spirilla  develop  in  the  inflamed 
intestinal  mucous  membrane,  but  cannot  grow  in  the  respiratory  tract, 
blood,  or  tissues.  The  tetanus  bacilli  develop  in  wounds  of  the  sub- 
cutaneous tissues,  but  cannot  grow  on  the  intestinal  mucous  mem- 
branes or  in  the  blood. 

Other  bacteria  find,  indeed,  certain  regions  especially  suitable  for 
their  growth,  but  under  conditions  favorable  for  them  are  capable  of 
developing  in  other  locations.  Thus,  the  typhoid  bacillus  grows 
most  luxuriantly  in  the  Peyer  patches  and  mesenteric  glands,  but  also 
invades  the  blood,  spleen,  and  other  regions.  The  tubercle  bacillus 
often  remains  localized  in  the  apex  of  a  lung  or  a  gland  for  years,  but 
at  any  time  may  invade  many  tissues  of  the  body.  The  gonococcus 
finds  the  mucous  membrane  of  the  genitourinary  tract  most  suitable 
for  its  development,  but  also  frequently  is  capable  of  growth  in  the 
peritoneum  and  even  sometimes  in  the  general  circulation.  The  pneu- 
mococcus  develops  most  readily  in  the  lungs,  but  also  invades  the  con- 
nective tissues,  serous  membranes,  and  the  blood. 

All  these  bacteria,  although  ordinarily  increasing  only  in  the  body  of 
man,  can  be  grown  on  suitable  dead  material. 

There  are  bacteria  which,  in  so  far  as  we  know,  find  the  bodies  of 
human  beings  or  animals  the  only  fit  soil  for  their  growth.  These  are 
strictly  the  true  parasites.  The  spirillum  of  relapsing  fever  grows  only 
in  man;  neither  the  food  nor  the  conditions  suitable  for  the  develop- 
ment of  this  micro-organism  outside  of  the  body  have  as  yet  been  dis- 
covered. 

Following  rather  closely  the  schematic  separation  of  bacteria  accord- 
ing to  their  relation  to  disease  we  might  classify  them  as : 

1.  Strict  saprophytes,  or  bacteria  which  grow  readily  in  suitable  dead 
organic  material,  but  not  in  the  body  under  ordinary  conditions. 

a.  Bacteria  which  in  their  growth  produce  no  substances  poisonous 
to  the  body,  which  are  capable  of  absorption  through  the  intestinal 
walls  or  act  on  its  epithelium. 

b.  Bacteria  which  produce  in  their  growth  in  dead  organic  matter 
poisons    capable  of    acting    on  the    mucous    membrane  or  of    being 
absorbed  into  the  animal  body. 


144 


PRINCIPLES  OF  BACTERIOLOGY 


2.  Parasites,    with   possibility   of   saprophytic   growth.      These    are 
bacteria  which  can  develop  either  as  parasites  or  saprophytes.     The 
different  varieties  vary  as  to  the  amount  of  poison  which  they  produce. 
Some  grow  luxuriantly  in  dead  organic  material   under  very  diverse 
conditions,  others  only  under  specially  favorable  conditions.     In  the 
body  they  also  vary — some  grow  extensively  in  the  blood,  while  others 
are  'limited  to  one  or  more  tissues,  some  being  widely  disseminated 
throughout  the  body,  while  others  are  localized  in  or  upon  a  certain 
portion  of  it. 

3.  Strict  parasites,  or  bacteria  which,  so  far  as  we  know,  grow  only 
in  the  living  animal  or  vegetable  organism.     These  again  vary  in  the 
amount  of  poison  which  they  produce  and  in  the  local  or  general  infec- 
tion to  which  they  give  rise. 

Adaptation  of  Bacteria  to  the  Soil  upon  which  They  Are  Grown. — Those 
bacteria  which  grow  both  in  living  and  dead  substances  vary  from  time 
to  time  as  to  their  readiness  to  develop  in  either  the  one  or  the  other. 
As  a  general  rule,  bacteria  grown  in  any  one  medium  become  more  and 
more  accustomed  to  that  and  other  media  more  or  less  analogous  to  it, 
while,  on  the  other  hand,  they  are  less  easily  cultivated  on  media  widely 
different  from  that  in  which  they  have  developed.  Thus  we  have  a 
culture  of  tubercle  bacilli  which,  after  having  grown  for  three  years  in 
the  bodies  of  guinea-pigs,  will  no  longer  develop  on  dead  organic  matter, 
while  a  bacillus  which  was  obtained  from  the  same  stock,  but  grown 
on  bouillon  for  three  years,  will  no  longer  increase  in  the  animal  body. 
From  the  same  stock,  therefore,  two  varieties  have  developed,  the  one 
being  now  practically  a  saprophyte  and  the  other  a  parasite. 

Local  Effects  Produced  by  Bacteria  and  Their  Products. — After  the  bac- 
teria gains  entrance  to  a  suitable  part  of  the  body  and  find  conditions 
favorable  for  growth  there  is  a  certain  lapse  of  time  before  sufficient 
bacterial  poisons  have  accumulated  to  cause  by  their  action  on  the  tissue 
noticeable  disturbance.  This  is  called  the  period  of  incubation.  Its 
length  depends  on  the  amount,  kind,  and  virulence  of  the  micro-organ- 
isms introduced  and  the  tissue  invaded.  The  incubation  period  over, 
we  note  the  course  of  the  local  and  general  lesions  excited  by  the  specific 
and  general  poisons.  The  extent  to  which  this  will  progress  depends, 
on  the  one  hand,  on  the  characteristics  of  the  invading  micro-organisms; 
on  the  other,  on  the  characteristics  of  the  tissues  invaded. 

The  local  effects  of  the  bacterial  poisons  upon  the  cells  give  rise  to 
the  various  kinds  of  inflammation,  such  as  serous,  fibrin ous,  purulent, 
croupous,  hemorrhagic,  necrotic,  and  gangrenous,  and  also  prolifera- 
tive,  as  seen,  for  example,  in  leprosy.  Some  bacteria  incite  specific 
forms  of  inflammation  along  with  those  common  to  many  bacteria; 
others  produce  no  peculiar  form  of  lesions. 

Thus  inflammation  and  serous  exudation  into  the  subcutaneous  tis- 
sues follow  injections  of  the  pneumococcus  or  anthrax  bacillus.  The 
development  of  the  streptococcus  or  pneumococcus  in  the  endocardium 
or  pleural  cavity  is  followed  by  a  serous  exudation,  frequently  with 
more  or  less  fibrin  production.  The  formation  of  pus  results  more 


THE  RELATION  OF  BACTKHIA    Tn  Dlsi:  \  j.- 

especially  from  the  streptococcus,  pneurnococcus,  and  staphylococcus ; 
but  nearly  all  forms  of  bacteria,  when  they  accumulate  in  one 
locality,  may  produce  purulent  inflammation.  The  colon,  typhoid, 
and  influenza  bacilli  frequently  cause  the  formation  of  abscesses. 

Catarrhal  inflammation,  with  or  without  pus,  follows  the  absorption 
of  the  products  of  many  bacteria,  such  as  the  gonococcus,  pneumo- 
coccus,  streptococcus,  and  influenza  bacillus,  etc.  The  hemorrhagic 
exudation  seen  in  pneumonia  is  usually  due  to  the  pneumococcus ;  it  is 
observed  also  in  other  infections.  Cell  necrosis  is  produced  frequently 
by  the  products  of  the  diphtheria  and  of  the  typhoid  bacilli  and  by 
those  of  other  bacteria.  Specific  proliferative  inflammation  follows 
the  localization  of  the  products  derived  from  the  tubercle  bacillus  and 
the  leprosy  bacillus. 

Not  only  can  the  poisons  of  one  species  of  bacteria,  according  to  the 
tissues  attacked,  produce  several  forms  of  inflammation,  but  the  same 
organism  will  vary  as  to  its  mode  and  extent  of  invasion;  this  depend- 
ing, first,  upon  its  own  characteristics  at  the  time  as  to  virulence,  etc., 
and,  second,  upon  the  conditions  in  the  infected  animal,  such  as  its 
health  and  power  of  resistance,  the  location  of  infection,  and  the  cir- 
cumstances under  which  the  animal  remains.  Such  variations,  there- 
fore, are  in  no  case  specific,  for  different  poisons  will  produce  changes 
which  appear  identical.  >, 

Manner  in  which  Bacteria  Produce  Injury. — The  actual  mechanical 
presence  of  the  bacteria  is  only  of  importance  when,  as  in  pronounced 
septicaemia  or  pyaemia,  they  exist  in  such  enormous  numbers  as  to  inter- 
fere mechanically  with  the  circulation  or  cause  minute  thrombi,  and 
later  emboli,  which  finally  produce  infarction  and  abscesses  in  different 
parts  of  the  body.  Even  these  dangerous  effects  are  almost  wholly 
due  to  the  chemical  substances  given  off,  which  are  more  or  less  directly 
poisonous.  Some  portion  of  the  protoplasm  of  almost  every  variety 
of  bacteria  acts  as  an  irritant  to  tissues  and  combines  with  some  of 
the  body  cells,  and  that  of  most  have  a  positive  chemotaxis. 

These  poisonous  products,  as  already  described  in  the  previous 
chapter,  can  be  separated  from  the  culture  fluid  in  which  the  bacteria 
have  grown  or  they  can  be  extracted  from  their  bodies.  These  prod- 
ucts without  the  bacteria  themselves  injected  into  animals  cause  essen- 
tially the  same  lesions  as  are  produced  by  the  bacteria  when  they  develop 
in  the  animal  body.  The  substances  contained  in  or  produced  by  the 
bacteria,  with  few  exceptions,  attract  the  leukocytes,  and  when  great 
masses  of  bacteria  die  suppuration  usually  follows. 

General  Symptoms  Caused  by  Bacterial  Poisons  Absorbed  into  the  Circu- 
lation.— Fever  is  produced  under  favorable  conditions  by  all  bacterial 
poisons.  The  first  requisite  is  that  sufficient  poisons  be  absorbed;  but, 
on  the  other  hand,  they  must  not  be  absorbed  with  such  rapidity  as 
to  overwhelm  the  infected,  for  a  moderate  dose  may  raise  the  tem- 
perature, while  a  very  large  dose  lowers  it,  as  occurs  sometimes  when 
a  very  large  surface,  such  as  the  peritoneum,  is  suddenly  involved.  The 
effect  of  fever  has  no  known  antibacterial  power,  but  it  may  be  due  to 

10 


146  PRINCIPLES  OF  BACTERIOLOGY 

some  part  of  the  reaction  of  the  tissues  which  in  other  portions  gives  rise 
to  the  antitoxins  and  antibactericidal  substances.  It  is  also  a  sign  that 
the  body  cells  as  a  whole  are  not  yet  overwhelmed  by  the  infection. 

With  few  exceptions  the  bacterial  poisons  produce  an  increase  in  the 
number  of  leukocytes  and  a  lessening  in  the  amount  of  haemoglobin 
in  the  blood.  In  uncomplicated  infection  with  typhoid  bacilli  there  is 
a  hypoleukocytosis.  The  different  varieties  of  leukocytes  are  increased 
in  varying  proportions  in  different  infections.  The  red-blood  cells  are 
directly  injured  by  a  number  of  bacterial  substances.  The  deleterious 
effects  on  the  nutrition  are  partly  due  to  the  direct  effect  of  the  poison 
and  partly  to  the  diseased  conditions  of  the  organs  of  the  body,  such  as 
the  spleen,  kidney,  and  liver.  Degeneration  of  the  nerve  cells  is  fre- 
quently noticed  after  infectious  diseases;  especially  is  this  true  of  diph- 
theria. Several  bacterial  poisons  have  been  found  to  produce  convul- 
sions; the  best  example  of  this  is  the  tetanus  toxin. 

Influence  of  Quantity  in  Infection. — With  pathogenic  bacteria  the 
number  introduced  has  an  immense  influence  upon  the  probability  of 
infection  taking  place. 

If  we  introduce  into  a  culture  medium  containing  some  fresh  human 
blood  or  serum  a  few  bacteria  it  is  probable  that  they  will  all  die;  whereas 
if  a  greater  number  are  introduced,  while  there  will  at  first  be  a  great 
diminution  of  these,  those  that  die,  having  combined  with  the  bacteri- 
cidal substances  in  the  serum,  neutralize  them ;  then  those  bacteria  which 
survive  begin  to  increase,  and  soon  they  multiply  enormously.  The 
same  is  true  for  parasitic  bacteria  in  the  body.  A  few  only  gaining 
entrance,  they  may  die;  a  larger  number  being  introduced,  some  may 
or  may  not  survive;  but  if  a  still  greater  quantity  is  injected  it  is  almost 
certain  that  there  will  be  some  surviving  members,  which,  because  of 
the  antagonistic  substances,  having  been  neutralized  or  of  their  having 
some  peculiar  properties,  will  begin  to  grow  and  excite  disease. 

With  those  bacteria  whose  virulence  is  great  a  very  few  organisms 
will  produce  disease  almost  as  quickly  as  a  million,  allowance  only 
being  made  for  the  short  time  required  for  the  few  to  become  equal  in 
number  to  the  million.  At  the  other  extreme  of  virulence,  however, 
many  millions  may  have  to  be  introduced  to  permit  of  the  development 
of  any  of  the  organisms  in  the  body.  With  these  bacteria  we  are  thus 
able  to  produce  either  no  effect  whatever,  a  local  effect,  or  in  some 
cases  a  general  septicaemia,  by  regulating  the  amount  of  infection  intro- 
duced. In  the  majority  of  cases  in  man  the  number  of  bacteria  re- 
ceived is  comparatively  small  ;  but  by  the  rupture  of  an  abscess  into 
a  body  cavity  or  into  the  circulation,  or  by  the  opening  of  the  intes- 
tinal contents  into  the.  peritoneum,  the  quantity  introduced  may  be 
enormous. 

Variation  in  Degree  of  Virulence  Possessed  by  Bacteria.— Bacteria 
differ,  as  has  already  been  stated,  as  to  the  ease  and  rapidity  with 
which  they  grow  in  any  nutritive  substance  and  the  amount  of  poison 
they  produce.  Both  of  these  properties  not  only  vary  greatly  in 
different  members  of  the  same  species,  but  each  variety  of  bacteria 


THE  RELATIOX  OF  BACTERIA  TO  DISEASE  147 

may  to  a  large  extent  be  increased  or  diminished  in  virulence.  The 
septicaemic  class  of  bacteria  when  grown  in  the  body  fluids  gradually 
produce  cells  with  less  substance  with  affinity  for  the  bactericidal  sub- 
stances of  the  blood  and  thus  become  less  vulnerable.  The  variation 
in  the  amount  of  specific  poisons  produced  by  bacteria  can  be  best 
studied  in  diphtheria  and  tetanus.  We  note,  first,  that  different 
cultures  of  diphtheria  and  tetanus  bacilli  have  wide  variations  in 
the  amount  of  toxin  which  they  produce — i.  e.,  a  diphtheria  bacillus 
obtained  from  a  case  of  diphtheria  will  produce  in  suitable  nutrient 
broth  a  poison  of  such  strength  that  1  c.c.  will  kill  an  average-sized 
guinea-pig,  while  the  poison  from  another  bacillus  will  kill  with  a 
much  less  quantity,  or  0.005  c.c.  Further,  the  bacilli  obtained  from 
some  sources  retain  their  power  of  producing  poison,  when  grown  on 
artificial  media,  for  years  unaltered,  while  others  lose  much  of  this  in 
a  few  months.  This  is  equally  true  of  the  tetanus  bacilli. 

The  power  to  produce  toxin  can  be  taken  from  bacilli  by  growing 
them  under  adverse  circumstances,  such  as  cultivation  at  the  maxi- 
mum temperature  at  which  they  are  capable  of  development.  Some 
bacilli  are  easily  attenuated;  others  are  robbed  of  their  virulence  only 
with  great  difficulty.  Increase  of  toxin-production  is  more  difficult, 
and  it  is  only  possible  to  obtain  it  to  a  certain  extent.  The  means 
usually  employed  are  the  frequent  replanting  of  cultures  But  with 
all  our  efforts  we  are  usually  only  able  to  restore  approximately  the 
degree  of  toxin-formation  which  the  cultures  originally  possessed.  The 
adaptation  of  bacteria  to  any  nutritive  substance,  living  or  dead,  so 
that  they  will  grow  more  readily,  is  more  easily  brought  about,  pro- 
vided they  will  grow  at  all.  The  streptococcus  from  erysipelas  and  the 
pneumococcus  from  pneumonia  are  typical  of  this  class  of  bacteria. 
Inoculate  a  rabbit  with  a  few  streptococci  obtained  from  a  case  of 
human  sepsis,  and,  as  a  rule,  no  result  follows;  inject  a  few  million, 
and  usually  a  local  induration  or  abscess  appears;  but  if  one  hundred 
million  are  administered  septicaemia  develops.  From  this  rabbit  now 
inoculate  another,  and  we  find  that  a  dose  slightly  smaller  suffices  to 
produce  the  same  effect;  in  the  next  animal  inoculated  from  this  still 
less  is  required,  and  so  on,  until  in  time,  with  some  cultures,  a  very 
minute  number  will  surely  develop  and  produce  death.  The  same 
increase  in  virulence  can  be  noted  when  septic  infection  is  carried  in 
surgery  or  obstetrics  from  one  human  case  to  another.  By  allowing 
bacteria  to  continue  to  develop  under  certain  fixed  conditions  they 
become  accustomed  to  them,  and  less  adapted  for  all  that  differ. 

Somewhat  distinct,  again,  from  that  class  of  bacteria  which  multiply 
rapidly  are  those  which,  like  the  tubercle  and  leprosy  bacilli,  because 
of  not  developing  in  the  blood  increase  more  slowly.  Here  increase  of 
virulence  is  shown,  as  before,  by  the  production  of  disease  through 
the  introduction  of  very  small  numbers  into  the  body,  but  increase 
in  rapidity  of  development  cannot  progress  except  to  within  certain 
limits.  A  single  streptococcus  may,  through  its  rapid  multiplication, 
produce  death  in  eighteen  hours;  a  single  tubercle  bacillus,  on  the 


148  PRINCIPLES  OF  BACTERIOLOGY 

other  hand,  cannot  produce  sufficient  numbers  in  less  than  two  weeks. 
The  virulence  of  the  septicaemic  class  of  bacteria  is  not  at  all  the  same 
when  measured  in  different  animals,  and  it  is  largely  for  this  reason 
that  the  virulence  in  test  animals  does  not  usually  correspond  with  the 
severity  of  the  case  from  which  the  organism  was  derived.  We  should 
remember  in  this  connection  the  varying  power  of  resistance  in  dif- 
ferent animals  to  the  same  organism  and  of  the  same  individual  at 
different  times. 

Mixed  Infection. — The  combined  effects  upon  the  tissues  of  the  prod- 
ucts of  two  or  more  varieties  of  pathogenic  bacteria,  and  also  of  the 
influence  of  these  different  forms  on  each  other,  are  of  great  impor- 
tance in  the  production  of  disease.  The  infection  from  several  different 
organisms  may  occur  at  the  same  time,  or  one  may  follow  the  other  or 
others — so-called  secondary  infection.  Thus,  an  abscess  is  often  due 
to  several  forms  of  pyogenic  cocci.  If  a  fresh  wound  is  infected  from 
such  a  source  the  inflammation  produced  will  probably  be  caused  by 
all  the  varieties  present  in  the  original  infection.  Peritonitis  following 
intestinal  injuries  must  necessarily  be  due  to  more  than  one  organism. 
Thus,  whenever  two  or  more  varieties  of  bacteria  are  transferred  to 
a  new  soil,  mixed  infection  takes  place  if  more  than  one  variety  is 
capable  of  developing  in  that  locality. 

Forms  of  infection  which  are  allied  to  both  mixed  and  secondary 
infection  are  those  occurring  in  the  mucous  membranes  of  the  respiratory 
and  digestive  tract.  In  these  situations  pathogenic  bacteria  of  slight 
virulence  are  always  present  even  in  health.  Thus,  in  the  upper  air 
passages  there  are  usually  found  streptococci,  staphylococci,  and  pneu- 
mococci.  When  through  a  cold,  or  the  invasion  of  another  infective 
agent,  as  the  diphtheria  bacillus,  the  virus  of  smallpox  or  scarlet  fever, 
the  epithelium  of  the  mucous  membrane  of  the  throat  is  injured  or 
destroyed,  the  pyogenic  cocci  already  present  are  now  enabled  in  this 
diseased  membrane  to  grow,  produce  their  poison,  and  even  invade 
deeper  tissues.  The  intestinal  mucous  membrane  is  invaded  in  a 
similar  way  by  the  colon  bacilli  and  other  organisms  after  injury  by 
the  typhoid  or  dysentery  bacilli  or  cholera  sgirilla.  Generally  speaking 
all  inflammations  of  the  mucous  membranes  and  skin  contain  some  of 
the  elements  of  mixed  infection.  Blood  infection,  on  the  other  hand, 
is  usually  due  to  one  form  of  bacteria,  as  even  when  several  varieties 
are  introduced,  only  one,  as  a  rule,  is  capable  of  development.  The 
same  is  true  to  a  somewhat  less  extent  of  inflammation  of  the  connec- 
tive tissue.  The  additional  poison  given  off  by  the  associated  bacteria 
aid  infection  by  the  primary  invaders  by  causing  a  lowering  of  the  vital 
resistance  of  the  body.  In  some  cases  the  secondary  infection  is  a 
greater  ^danger  than  the  primary  one,  as  pneumococcic  bronchopneu- 
monia  in  laryngeal  diphtheria  or  streptococcic  septicaemia  in  scarlet 
fever  and  smallpox. 

The  bacteria  are  also  at  times  directly  influenced  by  the  products 
of  associated  organisms.  These  may  affect  them  injuriously,  as,  for 
example,  the  pyogenic  cocci  in  anthrax;  or  they  may  be  necessary  to 


THE  RELATIOX  OF  BACTERIA  TO  DISEASE  149 

their  development,  as  in  the  case  of  anaerobic  bacteria.  Not  infrequently 
the  tetanus  bacilli  or  spores  would  not  be  able  to  develop  in  wounds 
were  it  not  for  the  presence  of  aerobic  bacteria  introduced  with  them. 
This  is  shown  outside  the  body,  where  tetanus  bacilli  will  not  grow  in 
the  presence  of  oxygen  unless  aerobic  bacteria  are  associated  with 
them.  Again,  it  is  found  that  the  association  of  one  variety  with  another 
mav  increase  its  virulence.  Streptococci  are  stated  to  increase  the  viru- 
lence of  diphtheria  bacilli,  but  here  it  is  probably  the  loss  of  resistance 
of  the  tissues  because  of  the  streptococcic  poison.  On  the  other  hand, 
the  absorption  of  the  products  of  certain  bacteria  immunizes  the  body 
against  the  invasion  of  other  bacteria,  as  shown  by  Pasteur  that 
attenuated  chicken-cholera  cultures  produce  immunity  against  anthrax. 

Ability  of  Bacteria  to  Penetrate  the  Skin  and  Mucous  Membranes.  THE 
SKIN. — The  unbroken  skin  is  a  great  protection  against  the  penetra- 
tion of  micro-organisms.  When  they  do  penetrate  it  is  through  the 
glands,  or  more  often  through  some  unobserved  wound.  Soluble 
vegetable  poisons,  such  as  aconite  and  bacterial  toxins,  are  not 
absorbed. 

There  is  an  apparent  exception  to  the  above  statements  in  the  fact 
that  the  pyogenic  staphylococci  and  sometimes  the  streptococci  exist 
upon  the  skin  or  in  it  between  its  superficial  horny  cells,  some  excep- 
tional circumstances,  such  as  wounds  or  burns,  being  required  to  allow 
the  organisms  to  penetrate  deeper.  The  cutaneous  sweat  glands,  and 
the  hair  follicles  with  their  appended  sebaceous  glands,  may  allow 
entrance  of  infection,  as  various  incidents  may  lead  to  the  introduction 
and  retention  of  virulent  micro-organisms.  When  this  occurs  the 
retained  products  may  lead  to  necrosis  of  the  epithelium  and  thus 
allow  the  bacteria  to  penetrate  to  the  deeper  tissues.  The  secretion 
of  the  sebaceous  glands  appears  not  to  be  bactericidal,  but  the  acidity 
of  the  perspiration  renders  it  slightly  so. 

SUBCUTANEOUS  CONNECTIVE  TISSUES. — Many  bacteria  cannot  de- 
velop in  the  connective  tissues  and  others  produce  a  milder  infection 
there  than  elsewhere.  Others  develop  readily  and  cause  infection.  The 
rapidity  of  development  of  new  connective  tissue  and  the  bactericidal 
properties  of  the  lymph  are  the  main  known  hindrances  to  infection. 

THE  Mucous  MEMBRANES. — The  moist  condition  of  the  surface  of 
the  membranes  aids  bacterial  multiplication.  Mucus  is  only  slightly 
bactericidal  for  some  bacteria  and  not  at  all  for  others.  Bacteria,  such 
as  the  pneumococci  and  streptococci,  remaining  in  it  become  somewhat 
attenuated.  The  conjunctival  mucous  membranes  are  protected  by 
the  cleansing  produced  by  the  flow  of  the  lacrymal  secretion  and  by 
its  slight  germicidal  action.  In  infancy  the  membranes  are  readily 
infected  by  gonococci  and  later  by  pneumococci,  the  Koch-Weeks 
bacillus  and  others.  Many  soluble  poisons  and  toxins  are  absorbed. 
The  mucous  membranes  of  the  nasal  cavity  are  somewhat  cleansed  by 
the  nasal  secretion,  which  is  feebly  bactericidal.  The  deeper  portions 
of  the  nasal  cavity  are  usually  the  seat  of  streptococci  and  other 
bacteria.  The  mouth  in  a  person  in  health  is  cleansed  by  the  feebly 


150  PRINCIPLES  OF  BACTERIOLOGY 

bactericidal  saliva.  When  the  teeth  are  decayed  many  varieties  of 
bacteria  abound.  The  bacteria,  such  as  the  diphtheria  bacilli,  strepto- 
cocci, etc.,  rarely  invade  the  mucous  membrane  of  the  tongue  or  mouth. 

The  tonsils  with  their  crypts  are  usually  the  seat  of  the  pyogenic 
cocci  and  are  readily  infected  by  the  diphtheria  bacilli  and  others. 
Whether  the  absolutely  intact  epithelium  allows  the  passage  of  these 
bacteria  is  disputed,  but  with  the  slight  pathological  lesions  usually 
present  it  undoubtedly  does. 

THE  LUNGS. — Most  inhaled  bacteria  which  pass  the  larynx  are 
caught  in  the  bronchi.  Many  of  these  are  gradually  removed  by  the 
ciliated  epithelium.  Both  the  alveolar  epithelial  cells  and  the  leukocytes 
which  enter  the  air  sacs  and  bronchioles  have  been  shown  to  take  up 
bacteria.  The  normal  lung  is,  therefore,  rapidly  freed  of  bacteria. 
Under  the  influences  of  certain  nervous  impressions  such  as  follow 
exposure  to  cold,  etc.,  certain  areas  of  the  lung  seem  to  lose  their 
protective  defences. 

THE  STOMACH. — The  pure  gastric  juice,  through  the  hydrochloric 
acid  it  contains,  is  able  to  kill  most  non-spore-bearing  organisms  in  a 
short  time,  but  because  of  neutralization  through  food,  or  because  the 
bacteria  are  protected  in  the  food,  many  of  them  pass  into  the  intestines. 
Tubercle,  typhoid,  colon,  and  dysentery  bacilli,  when  fed  by  the  mouth 
with  food,  readily  pass  through  the  stomach.  Certain  acidiphilic  germs, 
as  well  as  yeasts  and  torulse,  seem  to  grow  in  the  gastric  secretion;  these 
are  largely  non-pathogenic.  Perforation  of  the  stomach  is  usually 
followed  by  peritonitis,  because  of  the  irritant  effect  of  the  gastric  juice 
and  the  presence  of  bacteria  which  are  temporarily  retained.  The 
gastric  juice  neutralizes  tetanus  and  diphtheria  toxins.  Other  poisons, 
such  as  some  that  occur  in  decayed  meat,  are  not  neutralized.  The 
stomach  is  exceptionally  free  from  bacterial  inflammations. 

INTESTINES. — The  bile  is  feebly  bactericidal  for  some  bacteria,  but, 
on  the  whole,  the  intestinal  secretions  have  little  or  no  germicidal  power. 
The  number  of  bacteria  increase  steadily  from  the  duodenum  to  the 
head  of  the  colon,  and  diminish  slightly  from  the  upper  to  the  lower 
end  of  the  colon.  The  pancreatic  juice  destroys  many  of  the  toxic 
bacterial  products.  The  presence  of  the  bacilli  of  the  colon  group,  of 
streptococci,  etc.,  do  not  often  set  up  any  inflammatory  condition  in  the 
normal  intestines  of  healthy  persons.  In  children  suffering  from  the 
prostrating  effects  of  heat  they  are  apt  to  excite  inflammatory  changes. 
Even  pathogenic  bacteria,  such  as  the  typhoid,  dysentery,  and  tubercle 
bacilli,  may  pass  through  the  whole  length  of  the  healthy  intestines 
without  inciting  inflammations.  Slight  lesions  aid  the  passage  of  bacteria 
to  the  deeper  structures. 

Importance  of  Location  of  Point  of  Entry  of  Bacteria.  Most  bacteria 
cause  infection  only  when  they  gain  access  to  special  tissues  and  must, 
therefore,  enter  through  certain  portals.  This  fact  is  of  immense  impor- 
tance in  the  transmission  or  prevention  of  disease.  Thus,  for  example, 
let  us  rub  very  virulent  streptococci,  typhoid  bacilli,  and  diphtheria 
bacilli  into  an  abrasion  on  the  hand.  The  typhoid  bacillus  produces  no 


THE  REL.\TI<>\  OF  BACTERIA  TO  DISEASE  161 

lesion,  the  diphtheria  bacillus  but  a  very  minute  infected  area,  but  the 
streptococcus  may  give  rise  to  a  severe  cellulitis  or  fatal  septicaemia. 
Now  place  the  same  bacteria  on  an  abrasion  in  the  throat.  The 
typhoid  bacillus  is  again  harmless;  the  diphtheria  bacillus  produces 
inflammation,  a  pseudomembrane,  and  toxaemia,  and  the  streptococcus 
causes  an  exudate,  an  abscess,  or  a  septicaemia.  Finally,  introduce  the 
same  bacteria  into  the  intestines,  and  now  it  is  the  typhoid  bacillus 
which  produces  its  characteristic  lesions,  while  the  streptococcus  and 
diphtheria  bacillus  are  usually  innocuous. 

If  we  tried  in  this  way  all  the  parasitic  bacteria  we  would  find  that 
certain  varieties  are  capable  of  developing  and  thereby  exciting  dis- 
ease only  on  the  mucous  membrane  of  the  throat,  others  of  the  intes- 
tine, others  of  the  urethra;  some  develop  only  in  the  connective  tissues 
or  in  the  blood,  while  others,  again,  under  favorable  conditions,  seem 
able  to  grow  in  or  upon  most  regions  of  the  body. 

The  Dissemination  of  Disease. — The  spread  of  infection  is  influenced 
by:  1.  The  number  of  species  of  animals  subject  to  infection. 

Many  human  infectious  diseases  do  not  occur  in  animals,  and  many 
animal  infections  are  not  found  in  man.  Thus,  so  far  as  we  know, 
gonorrhoea,  syphilis,  measles,  smallpox,  typhoid  fever,  etc.,  do  not 
occur  in  animals  under  ordinary  conditions;  while  tuberculosis,  anthrax, 
glanders,  hydrophobia,  and  some  other  diseases  are  common  to  both 
man  and  animals. 

2.  The  quantity  of  the  infectious  material  and  the  manner  in  which 
it  is  thrown  off  from  the  body. 

In  diphtheria,  typhoid  fever,  cholera,  pulmonary  tuberculosis,  septic 
endometritis,  influenza,  and  gonorrhoea  enormous  numbers  of  infec- 
tious bacteria  are  cast  off  through  the  discharges  from  the  mouth, 
intestines,  and  genitourinary  secretions,  causing  great  danger  of  infec- 
tion. On  the  other  hand,  in  tuberculous  peritonitis,  relapsing  fever, 
septic  pleurisy  and  endocarditis,  gonorrhreal  rheumatism,  and  the  like, 
there  is  little  or  no  danger  of  infecting  others,  as  few  or  no  bacteria  are 
cast  off. 

3.  The  resistance  of  the  infectious  bacteria  to  the  deleterious  effects 
of  drying,  light,  heat,  etc. 

In  this  case  the  presence  or  absence  of  spores  is  of  the  greatest  impor- 
tance. The  spore-bearing  bacilli,  such  as  tetanus,  anthrax,  etc.,  being 
able  to  withstand  destruction  for  a  long  time,  retain  their  power  of 
producing  infection  for  months  or  even  years  after  elimination  from 
the  body.  The  bacteria  which  form  no  spores  show  great  variation  in 
their  resistance  to  outside  influences.  Some  of  these,  such  as  the  influ- 
enza bacilli  and  the  gonococci,  the  virus  of  syphilis  and  hydrophobia, 
are  extremely  sensitive;  the  pneumococci,  cholera  spirilla,  glanders 
bacilli,  etc.,  are  a  little  hardier;  then  follow  the  diphtheria  bacilli, 
and  after  them  the  typhoid  and  tubercle  bacilli  and  the  staphylo- 
cocci. 

4.  The  ability  or  the  lack  of  ability  to  grow  outside  of  the  infected 
tissues. 


PRINCIPLES  OF  BACTERIOLOGY 

Such  bacteria  as  the  pneumococcus,  tubercle,  influenza,  diphtheria, 
glanders,  and  leprosy  bacilli  do  not  develop,  as  far  as  we  know,  out- 
side of  the  body  under  ordinary  conditions.  Under  exceptional  circum- 
stances, as  in  milk,  some  may  develop.  Others,  again,  such  as  the 
streptococcus  and  staphylococcus,  typhoid  and  anthrax  bacillus,  the 
cholera  spirillum,  and  some  anaerobics,  may  develop  under  peculiar 
conditions  existing  in  water  or  soil. 

While  for  the  pathogenic  bacteria,  as  a  rule,  the  saprophytes  met 
with  in  the  soil  and  water  are  antagonistic,  yet  in  some  cases — and 
especially  is  this  true  of  the  anaerobic  bacteria — they  are  helpful. 
Such  bacilli  as  tetanus  are  believed  to  require  the  association  of 
anaerobic  bacteria  to  permit  of  their  development  in  the  soil  in  the 
presence  of  oxygen. 

5.  Ability  to  develop  in  or  upon  some  portion  of  the  skin  or  mucous 
membrane,  either  after  or  before  disease,  and  without  causing  infection. 
As  complete  a  knowledge  as  possible  of  this  saprophytic  development  in 
man  of  parasitic  bacteria  is  necessary  if  we  are  to  combat  the  spread 
of  infection.  In  the  superficial  layers  of  the  epithelium  and  on  the  sur- 
face of  the  skin  we  find  the  different  pyogenic  cocci,  which  are  capable 
of  infecting  a  wounded  or  injured  part  or  causing  inflammation  in  the 
glands.  Acne,  the  pustules  in  smallpox,  the  pus  on  a  burned  surface, 
boils,  etc.,  all  come  from  these  pyogenic  cocci.  In  surgical  cases  the 
skin  has  to  be  as  thoroughly  disinfected  as  possible,  to  prevent  the 
formation  of  stitch-hole  abscesses  and  wound  suppuration. 

In  the  secretion  of  the  mucous  membrane  covering  the  pharynx  and 
nasopharynx  there  is  always  an  abundance  of  bacteria.  In  throats  ex- 
amined in  New  York  City,  streptococci,  staphylococci,  and  pneumo- 
cocci  are  found  in  almost  every  instance,  and  even  in  the  country  they 
are  often  present.  In  the  anterior  nares  there  are  fewer  parasitic 
bacteria  than  in  the  posterior  portions.  The  nasal  secretion  is  only 
very  slightly,  if  at  all,  bactericidal.  Many  other  varieties  of  bacteria, 
such  as  the  meningococci  and  the  influenza  bacilli,  are  probably  often 
present  in  small  numbers.  In  those  constantly  in  contact  with  cases 
of  diphtheria,  and  in  those  convalescent  from  diphtheria,  virulent 
diphtheria  bacilli  are  frequently  found  in  the  throat. 

After  exposure  to  cold  or  injury  of  any  kind,  owing  to  the  presence 
of  these  bacteria,  the  persons  harboring  them  may  develop  tonsillitis, 
tonsillar  abscess,  or  diphtheria;  or  the  bacteria  may  invade  the  bron- 
chial mucous  membrane  or  the  lungs.  The  diphtheria  bacilli,  and 
perhaps  other  bacteria,  transmitted  to  others  may  become  the  source 
of  infection  to  them,  though  the  person  who  spreads  the  infection  may 
remain  unaffected. 

After  typhoid  fever  the  bacilli  may  remain  in  the  intestinal  con- 
tents for  weeks  and  in  the  bladder  and  gall-bladder  for  months. 

Autoinfection. — Some  good  observers  have  stated  that  bacteria  can 
be  absorbed  through  the  intestinal  wall  into  the  chyle  and  blood. 
When  the  intestinal  canal  is  injured,  or  its  circulation  hindered  by 
strangulation,  etc.,  the  bacillus  coli  and  some  other  bacteria  may 


777 /•:  RELATION  or  BACTERIA   TO  DISEASE 

penetrate  through  the  injured  walls  and  cause  peritonitis  or  general 
infection.  Under  certain  conditions,  as  during  the  debility  due  to  hot 
weather,  the  bacteria  in  the  intestines  cause,  through  their  products, 
irritation,  and  in  children  even  serious  intestinal  inflammation. 

The  kidneys,  bladder,  and  urethra  may  be  the  source  of  infection 
and  may  give  rise  to  disease.  Long  after  an  acute  gonorrhoea  has 
passed  gonococci  may  remain  in  sufficient  numbers  to  cause  a  new 
inflammation  or  produce  infection  in  others.  A  cystitis  may  run  on 
chronically  for  years,  and  then  suddenly  become  acute  or  spread  infec- 
tion to  the  kidneys.  A  persistent  gonorrhceal  vaginal  infection  may 
lead  to  a  gonorrhoeal  endometritis,  or  salpingitis,  or  peritonitis,  under 
suitable  conditions.  The  staphylococci  in  the  skin  and  the  colon 
bacilli  and  pyogenic  cocci  in  the  fecal  discharges  may  also  be  carried 
into  the  bladder  and  uterus  and  produce  septic  infection. 

Occasionally  the  casting  off  of  the  bacteria  allows  them  to  infect 
other  places,  as  in  some  cases  where  laryngeal  and  intestinal  tuber- 
culosis follow  pulmonary  tuberculosis.  We  must  bear  in  mind,  how- 
ever, that  infection  in  these  regions  may  have  been  produced  through 
the  lymph-  and  blood-channels. 

In  nearly  all  cases  of  infection  the  products  of  bacterial  growth  are 
absorbed  into  the  blood,  and  along  with  them  a  few  bacteria  also,  even 
when  they  do  not  reproduce  themselves  in  it.  The  greater  the  extent 
of  the  infection  and  the  more  deep  seated  it  is,  the  greater  is  the  amount 
of  absorption.  The  bacteria  enter  the  blood,  according  to  Kruse,  by 
(1)  passive  entrance  through  the  stromata  of  the  capillary  walls;  (2) 
carriage  into  the  blood  in  the  bodies  of  leukocytes;  (3)  growth  of  the 
bacteria  through  the  walls  of  the  vessels;  (4)  transmission  of  the  bac- 
teria through  the  lymph  glands  placed  between  the  lymph-  and  blood- 
vessels. 

When  bacteria  are  abundant  in  the  blood  they  become  fixed  in  the 
capillaries  of  one  or  all  of  the  organs,  especilly  of  the  liver,  kidneys, 
spleen,  and  lungs,  and  then,  by  means  of  the  leukocytes,  which  pene- 
trate the  capillary  walls,  or,  directly,  they  pass  into  the  tissues  and 
substance  of  the  organs.  They  thus  reach  the  lymph  channels  and 
glands,  or  through  the  secretions  gain  entrance  into  the  gall-bladder, 
saliva,  etc.,  or  press  through  the  epithelium,  as  in  the  alveoli  of  the 
lungs;  more  rarely  they  pass  through  the  excretions  into  the  urine,  as 
in  typhoid  fever,  though  some  deny  that  this  can  happen  unless  there 
is  a  previous  inflammation  of  the  kidneys. 

Elimination  of  Bacteria  through  the  Milk. — The  passage  of  bacteria 
through  the  breast  is  important,  from  the  fact  that  milk  is  so  largely 
used  as  food.  Many  observers  have  reported  the  finding  of  tubercle 
bacilli  in  milk  when  the  gland  itself  was  intact  and  the  animal  tuber- 
culous. Some  authorities  have  put  its  presence  in  milk,  under  these 
circumstances,  as  high  as  50  per  cent,  of  the  cases.  This,  in  our  experi- 
ence, is  undoubtedly  too  high,  and  probably  these  observers  have  been 
deceived  by  the  pseudotubercle  bacilli.  They  are  undoubtedly  present, 
however,  in  the  milk  of  some  animals  in  which  tuberculous  disease  of 


154  PRINCIPLES  OF  BACTERIOLOGY 

the  gland  could  not  be  demonstrated.  The  finding  of  streptococci  and 
staphylococci  is  due  probably  in  the  majority  of  cases  to  the  infec- 
tions taking  place  as  the  milk  is  voided,  for  the  epithelium  at  the  outlet 
of  the  lacteal  ducts  is  always  infected  with  staphylococci,  and  usually 
streptococci,  which  have  often  been  received  from  the  mouth  of  the 
sucking  infant. 

Elimination  of  Bacteria  by  the  Skin  and  Mucous  Membranes. — Whether 
bacteria  pass  from  the  blood  by  the  sweat  is  a  mooted  point.  The 
skin  is  always  the  seat  of  the  staphylococcus  and  frequently  of  other 
bacteria,  so  that  it  is  difficult  to  determine  in  any  given  case  the 
origin  of  the  bacteria  found  in  the  sweat.  Many  observers  have 
reported  the  passage  of  bacteria  from  the  blood  through  the  mucous 
membrane.  So  long  as  the  organs  of  secretion  are  not  injured  it  is  not 
likely  that  many  micro-organisms  are  eliminated  from  the  blood  in 
this  way.  Bacteria  are  sometimes  eliminated  through  the  urine,  but 
here,  as  a  rule,  when  great  numbers  of  organisms  are  found,  it  is  due  to 
development  in  the  bladder.  Such  removal,  moreover,  has  little  if  any 
beneficial  effect;  but,  on  the  other  hand,  it  may  be  a  source  of  danger 
to  others,  as  in  typhoid  fever.  The  removal  of  the  poisonous  products 
of  bacteria  by  the  kindeys,  intestines,  etc.,  on  the  contrary,  is  of  great 
advantage  to  the  organism. 


CHAPTER   XIV. 

THE  ANTAGONISM  EXISTING  BETWEEN  THE  LIVING  BODY 
AND  MICRO-ORGANISMS. 

THAT  certain  races  of  animals  and  men,  and  certain  individuals 
among  these,  are  more  refractory  to  disease  than  others  is  a  fact  which 
has  long  been  known.  Experience  and  observation  have  taught  us, 
further,  that  the  same  individuals  are  at  one  time  more  resistant  to 
disease  than  at  another.  This  inborn  or  spontaneous  refractory  con- 
dition to  an  infectious  disease  is  termed  natural  immunity,  in  contra- 
distinction to  that  acquired  by  recovery  from  the  disease. 

In  bacteria,  we  distinguish  between  the  ability  to  produce  poison 
and  the  power  to  multiply  in  the  body.  In  animals  and  the  higher 
plants  we  distinguish  between  immunity  to  poison  and  power  to 
inhibit  the  development  of  bacteria. 

In  regard  to  variations  in  susceptibility,  certain  known  facts  have 
been  accumulated.  Thus,  cold-blooded  animals  are  generally  insuscep- 
tible to  infection  from  those  bacteria  which  produce  disease  in  warm- 
blooded animals,  and  vice  versa.  This  is  partly  explained  by  the  ina- 
bility of  the  bacteria  which  grow  at  the  temperature  of  warm-blooded 
animals  to  thrive  at  the  temperature  commonly  existing  in  cold-blooded 
animals.  But  differences  are  observed  not  only  between  warm-blooded 
and  cold-blooded  animals,  but  also  between  the  several  races  of  warm- 
blooded animals.  The  anthrax  bacillus  is  very  infectious  for  the  mouse 
and  guinea-pig,  while  the  rat  is  not  susceptible  to  it  unless  its  body 
resistance  is  reduced  by  disease  and  the  amount  of  infection  is  great. 
The  inability  of  the  micro-organism  to  grow  in  the  body  of  an  animal 
does  not  usually  indicate,  however,  an  insusceptibility  to  its  poison; 
thus,  for  instance,  rabbits  are  less  susceptible  than  dogs  to  the  effects 
of  the  poison  elaborated  by  the  pneumococci,  but  these  bacteria  develop 
much  better  in  the  former  than  in  the  latter.  Differences  in  suscepti- 
bility are  sometimes  very  marked  among  different  varieties  of  the  same 
race  of  animals,  as,  for  instance,  between  different  kinds  of  rats  and 
pigeons  to  anthrax.  In  animals,  as  a  whole,  it  is  noticed  experimentally 
that  the  young  of  all  species  are  less  resistant  to  infection  than  the  older 
and  larger  ones. 

The  difficulty  experienced  by  the  large  majority  of  bacteria  in  devel- 
oping in  the  tissues  of  the  healthy  body  can  be  to  a  great  extent  removed 
by  any  cause  which  lowers  the  general  or  local  vitality  of  the  tissues. 
Among  the  causes  which  bring  about  such  lessened  resistance  of  the 
body  are  hunger  and  starvation,  bad  ventilation  and  heating,  exhaustion 
from  overexertion,  exposure  to  cold,  the  deleterious  effects  of  poisons, 


PRINCIPLES  OF  BACTERIOLOGY 

bacterial  or  other,  acute  and  chronic  diseases,  vicious  habits,  drunken- 
ness, etc.  Purely  local  injuries,  such  as  wounds,  contusions,  etc..,  give 
a  point  of  entrance  for  infection,  and  a  point  of  less  resistance,  where 
the  bacteria  may  develop  and  produce  local  inflammation.  Local 
affections,  such  as  endocarditis,  may  also  afford  an  area  of  lessened 
resistance  for  the  bacteria  to  seize  upon.  The  presence  of  foreign 
bodies  in  the  tissues  in  like  manner  predisposes  them  to  bacterial 
invasion.  Interference  with  free  circulation  of  blood  and  retention  in 
the  body  of  poisonous  substances  which  should  be  eliminated  also  tend 
to  lessen  the  vitality.  In  these  and  other  similar  ways  animals  which 
are  otherwise  refractory  may  acquire  a  susceptibility  to  disease. 

Increase  of  Resistance  by  Non-specific  Means. — Just  as  all  conditions 
which  are  deleterious  to  the  body  lessen  its  power  of  resistance  to 
bacterial  invasion,  so  all  conditions  which  are  favorable  to  it  increase, 
its  resistance,  and  thus  aid  in  preventing  and  overcoming  infection. 
The  internal  use  of  antiseptics  against  bacteria  has  not  proved  success- 
ful, for  the  reason  that  an  amount  too  small  to  inhibit  bacterial  growth 
is  found  to  be  poisonous  to  the  tissue  cells.  The  efficacy  of  quinine  in 
malaria  and  mercury  in  syphilis  is,  possibly,  an  exception  to  the  rule, 
but  in  both  cases  we  are  dealing  probably  with  animal  parasites,  not 
with  true  bacteria.  Such  substances  as  nuclein  and  others  contained 
in  blood  serum,  when  introduced  into  the  body  in  considerable  quan- 
tity, aid  somewhat  in  inhibiting  or  preventing  the  growth  of  many 
bacteria.  Even  bouillon,  salt  solution,  and  small  amounts  of  urine 
have  a  slight  inhibitory  action.  The  hastening  of  elimination  of  the 
bacterial  poisons  by  free  intestinal  evacuation  and  encouragement  of 
the  functions  of  the  skin  and  kidneys  are  also  of  some  avail.  The 
enzymes  formed  by  certain  bacteria  have  been  found  to  exert  a  slight 
bactericidal  action  not  only  on  the  germs  which  have  directly  or  indi- 
rectly produced  them  in  the  body,  but  also  on  other  varieties.  None 
of  these  enzymes  are  sufficiently  protective  to  be  of  practical  value  nor 
equal  in  power  to  the  protective  substances  formed  by  the  tissues  from 
the  bacterial  products. 

Use  of  Local  Treatment  in  Limiting  Bacterial  Invasion. — The  total 
extirpation .  of  the  infected  area  by  surgical  means,  if  thoroughly 
carried  out,  removes  the  bacteria  entirely;  but,  unfortunately,  this  pro- 
cedure is  rarely  possible.  When  incomplete  it  is  frequently  helpful; 
but  it  may  be  harmful,  for  by  creating  tissue  injury  and  exposing  fresh 
wounded  surfaces  to  infecion  it  may  lead  to  the  further  development  of 
the  disease.  Again,  it  is  usually  insufficient,  for  by  removing  only  a 
portion  of  the  bacteria  it  may  leave  those  which  have  already  reached 
the  deeper  tissues  or  blood  to  go  on  developing.  In  some  cases,  like 
anthrax  and  infection  from  bites  of  rabid  animals,  total  or  almost  com- 
plete removal  of  the  virus  is  possible,  either  by  the  knife  or  thorough 
cauterization,  and  will  prevent  a  general  infection  or  so  lessen  the  num- 
ber of  bacteria  in  the  body  as  to  allow  the  bactericidal  elements  of  its 
fluids  to  exterminate  them.  So  also  in  tetanus,  the  invasion  being 
limited,  surgical  interference  may  be  of  great  use  by  removing  not  only 


.'l.\T.U/n.Y/.S.U  />'/:TirAV-:.Y  THE  LlVIXd  HOI>Y  AM)  HACTERIA     157 

the  bacilli  themselves,  but  also  that  portion  of  their  poison  which  has 
not  as  yet  been  absorbed  from  the  tissues.  The  beneficial  effects  of 
opening  an  abscess,  incising  a  cellulitis,  or  cleansing  and  drainage  of 
the  uterine  cavity  are  well  known.  The  retention  of  the  poisonous 
products  of  the  bacteria  leads  to  their  absorption,  and  then  through 
their  combining  with  the  protective  substances  of  the  adjacent  fluids 
the  tone  of  the  neighboring,  and  to  a  less  extent  of  the  general,  tissues 
is  lowered.  This  enables  the  bacteria  to  penetrate  into  tissues  which 
would  otherwise  resist  them.  The  mechanical  effect  of  pressure  on 
the  walls  of  an  abscess  by  its  contents  also  aids  the  bacterial  progress. 
Local  bleeding  and  the  application  of  cold  probably  act  by  lessening 
tension.  The  application  of  warmth  increases  the  blood  flow  to  the 
part,  and  so,  when  the  general  blood  supply  is  bactericidal,  as  it  often 
is,  it  acts  favorably  on  the  inflammation  by  destroying  the  bacteria. 
A  peculiar  effect  of  operative  interference  is  noticed  in  the  frequently 
observed  beneficial  result  of  laparotomy  in  tuberculous  peritonitis. 

Antiseptic  solutions  have  the  power  of  cleansing  and  rendering  sterile 
the  surfaces  of  a  wound — that  is,  of  preventing  the  introduction  of 
infection.  After  infection  has  taken  place,  however,  it  is  doubtful 
whether  antiseptic  washing  has  much  more  direct  influence  than  simple 
cleansing,  and  it  certainly  can  have  no  bactericidal  effect  at  any  dis- 
tance from  the  surface,  either  direct  or  indirect.  Certain  infectious 
diseases  which  are  comparatively  superficial  are  probably  benefited 
by  antiseptic  solutions,  such  as  gonorrhoea,  diphtheria,  and  other 
inflammations  of  the  mucous  membranes.  Even  here,  however,  it  is 
impossible  to  do  more  than  disinfect  superficially,  and  in  some  cases 
any  irritation  of  the  tissues  is  apt  to  do  more  harm  than  good.  In  the 
superficial  lesions  of  syphilis  and  tuberculosis  the  local  use  of  antiseptics 
is  sometimes  of  great  value.  In  these  diseases  the  irritant  effects  of  the 
antiseptics  which  stimulate  the  tissues  may  also  be  beneficial. 

Specific  Immunity,  or  a  Condition  of  the  Body  which  Prevents  the  Develop- 
ment in  it  of  One  Variety  of  Micro-organisms  or  Renders  it  Unaffected 
by  Their  Bacterial  Poisons. — The  invasion  of  the  body  with  more  or  less 
serious  results  by  almost  every  micro-organism  is  followed  by  a  condition 
which  for  a  variable  period  and  to  a  variable  degree  is  deleterious  to  its 
further  growth.  It  also  may  give  rise  to  substances  which  neutralize 
the  poisonous  effects  of  the  bacterial  products.  This  specific  immunity 
may  take  place  in  various  ways: 

1.  Through    recovery   from   disease    naturally   contracted    or   from 
infection  artificially  produced.    According  to  the  nature  of  the  invading 
micro-organism  this  immunity  may  be  slight,  as  after  recovery  from 
erysipelas  or  pneumonia,  marked  for  a  short  period  of  time,  as  in  diph- 
theria and  typhoid  fever,  or  prolonged,  as  after  scarlet  fever  or  syphilis. 

2.  By  the  injection  of  micro-organisms  attenuated  by  heat,  chemi- 
cals, or  other  means.    In  this  case  an  infection  of  the  animal  is  produced, 
of  moderate  severity,  as  a  rule,  and  the  immunity  is  not  as  marked  and 
lasting  as  after  recovery  from  a  more  serious  attack;  but  it  is,  never- 
theless, considerable.     The  inoculation  of  sheep  with  the  attenuated 


158  PRINCIPLES  OF  BACTERIOLOGY 

anthrax  bacillus  and  the  use  of  vaccination  in  man  are  examples  of 
this  method. 

3.  By  the  injection  of  the  organisms  into  tissues  where  development 
will  not  take  place,  as  the  injection  of  typhoid  bacilli  or  cholera  spirilla 
into  the  subcutaneous  tissues.     Here  the  solution  of  the  bacteria  with 
the  absorption  of  their  products  causes  a  mild  chemical  poisoning,  with 
considerable  resulting  immunity. 

4.  By  the  injection  of  the  chemical  constituents  of  the  dead  bodies 
of  bacteria  and   of  the  chemical  products  which  they  elaborate  and 
discharge  into  the  surrounding  culture  media  during  their  life.    Men 
as  well  as  animals  have  been  immunized   by  the  injections  of   dead 
cultures  of   typhoid  and  anthrax  bacilli  and  cholera  spirilla,  etc.     A 
few  days  after  infection  with  most  parasitic  bacteria  the  body  resistance 
to  the  growth  of  the  same  organism   is    greatly  increased;   in    other 
infections,    however,   it  is  but  slightly  augmented.      This    increased 
resistance  is  readily  shown  to  be  partly  due  to  protective  substances 
held  in  solution  in  the  blood-serum  and  partly  to  the  leukocytes. 

5.  By  the  injection  of  the  blood  serum  of  animals  which  have  pre- 
viously passed  through  a  specific  disease  or  have  been  inoculated  with 
the  bacterial  products.    The  first,  probably,  to  think  of  the  possibility 
of  effecting  this  was  Raynaud,  who  in  1877  showed  that  the  injection 
of  large  quantities  of  serum  derived  from  a  vaccinated  calf  into  an 
animal  prevented  its  successful  vaccination.     Hericourt,  Richet,  and 
others  demonstrated  the  same  thing  for  other  diseases.     The  results 
obtained  by  Behring  and  Kitasato  upon  diphtheria  and  tetanus,  where, 
indeed,  the  serum  prevented  the  action  of  the  poisons  rather  than  the 
direct  development  of  the  bacteria,  gave  a  still  greater  impetus  to  these 
investigations. 

Suitable  animals  after  repeated  infections  gradually  accumulate  in 
their  blood  considerable  amounts  of  these  protective  substances,  so 
that  very  small  amounts  of  serum  inserted  in  another  animal  will  inhibit 
the  growth  of  the  bacteria  or  neutralize  their  products.  Thus,  0.1  c.c. 
of  a  serum  from  a  horse  frequently  infected  by  the  pneumococcus  will 
prevent  the  development  in  the  body  of  a  rabbit  of  one  hundred  times 
the  fatal  dose  of  very  virulent  pneumococci,  and  a  few  times  a  fatal 
dose  of  less  virulent  ones,  the  actual  number  as  well  as  the  virulence 
of  the  bacteria  affecting  the  protective  value  of  the  serum. 

These  protective  substances  are  found  also  in  other  fluids  of  the  body 
than  in  the  blood;  they  occur,  indeed,  in  the  substance  of  many  cells 
to  a  greater  or  less  extent. 

The  immunity  produced  by  these  five  methods  affects  the  entire  body, 
as  is  natural,  since  the  blood  into  which  they  are  absorbed  is  dis- 
tributed everywhere.  When  the  immunity  is  but  slight,  infection  may 
take  place  in  the  more  sensitive  regions,  and  still  be  impossible  in  those 
tissues  having  more  natural  resistance. 

Passive  as  Contrasted  with  Active  Immunity. — If  the  serum  is  injected 
into  animals  or  man  the  immunity  is  greatest  immediately  after  ab- 
sorption, and  then  declines,  being  rather  quickly  (in  several  weeks  or 


.\.\TA(;u\isM  IU-:T\VI-:I-:\  TIII-:  uvTm  BODY  AND  BACTERIA    159 

months)  almost  entirely  lost,  so  that  repeated  injections  are  required 
to  maintain  the  immunity.  This  passive  immunity  is  distinctly  in 
contrast  to  the  active  immunity  acquired  after  the  introduction  of 
bacterial  products,  where  the  tissues  of  the  organism,  in  ways  to  us 
unknown,  throw  out,  in  response  to  the  bacterial  stimulus,  inhibitory 
or  antitoxic  substances,  or  combine  with  the  bacterial  poisons  to 
produce  them.  Here  immunity  is  actually  lessened  for  one  or  two 
days,  and  then  is  increased,  and  reaches  its  height  a  week  or  ten  days 
after  the  injection,  and  then  continues  for  a  week  or  two,  when 
it  slowly  declines  again.  The  immunity  produced  by  the  transfer 
of  serum  from  the  immunized  to  the  non-immunized  is  frequently 
called  passive  immunity  and  the  immunity  produced  by  infection 
active  immunity. 

Production  of  Antitoxin  for  Therapeutic  Purposes. — If  a  greater 
quantity  of  protective  substance  than  is  required  for  the  protection 
of  the  individual  is  desired  in  the  blood,  repeated  injections  of  living 
or  dead  bacteria  and  their  products  are  given,  the  doses  being  ad- 
ministered at  short  intervals,  and  in  sufficient  amount  to  produce  a 
slight  elevation  of  temperature  and  malaise.  After  several  months  of 
such  treatment  the  blood  is  withdrawn,  allowed  to  clot,  and  the  serum 
then  siphoned  off  aseptically  and  stored  either  with  or  without  the  addi- 
tion of  preservatives. 

Testing  of  Protective  Power  of  Antibacterial  and  Antitoxic  Sera.— 
The  serum  is  tested  by  mixing  it  with  a  certain  number  of  times 
the  fatal  dose  of  a  culture  or  its  toxins  whose  virulence  or  toxicity 
is  known,  and  then  injecting  this  under  the  skin,  in  the  vein,  or 
into  the  peritoneum,  according  to  the  nature  of  the  bacteria  to  be 
tested.  The  main  point  is  that  some  definite  method  be  carried  out 
by  which  the  relative  value  of  the  serum  can  be  judged  in  comparison 
with  other  serums.  As  a  rule,  the  value  is  stated  in  the  number  of 
fatal  doses  of  culture  or  toxin  which  a  fraction  of  a  cubic  centimetre  of 
serum  will  prevent  from  destroying  the  animal.  It  is  well  to  remember 
that  with  a  living  germ  a  multiple  of  a  fatal  dose  is  not  as  much  more 
severe  than  a  single  dose  as  the  figure  would  suggest.  One  thousand 
times  a  fatal  dose  of  a  very  virulent  micro-organism  will  be  neutralized 
by  several  times  the  amount  of  serum  which  a  single  fatal  dose  requires, 
since  in  the  case  of  very  virulent  living  bacteria,  whose  virulence  is  due 
to  their  ability  to  increase,  it  is  not  the  organisms  which  are  introduced 
that  kill,  but  the  millions  that  develop  from  them. 

Limitation  of  Curative  Power  of  Serums. — As  a  rule,  the  serum  has  to 
be  given  before  the  bacteria  introduced  into  the  body  have  multiplied 
greatly.  After  that  period  has  elapsed  the  serum  usually  fails  to  act. 
This  is  due  to  the  fact  that  bactericidal  power  in  serum  is  due  to 
the  combined  effect  of  two  substances,  one  only  being  contained  in  the 
injected  serum.  The  serum  loses  all  appreciable  protective  value  as 
measured  in  test  animals  in  the  usual  doses  before  the  person  is 
liable  to  infection.  Repeated  injections  of  serum  continue  this  con- 
dition of  immunity  indefinitely. 


160  PRINCIPLES  OF  BACTERIOLOGY 

Practical  Value  of  Injections  of  Bactericidal  Sera. — The  use  of  serums 
having  specific  protective  properties  has  been  tried  practically  on  a  large 
scale  in  man  as  a  preventive  of  infection.  In  susceptible  animals 
injections  of  some  of  the  very  virulent  bacteria,  as  pneumococci, 
streptococci,  typhoid  bacilli,  and  cholera  spirilla,  can  be  robbed  of  all 
danger  if  small  doses  of  their  respective  serums  are  given  before  the 
bacteria  have  increased  to  any  great  extent  in  the  body.  If  given 
later  they  are  ineffective.  For  some  bacteria,  such  as  tubercle  bacilli,  no 
serum  has  been  obtained  of  sufficient  power  to  surely  prevent  infection. 
Through  bactericidal  serums,  therefore,  we  can  immunize  against  an 
infection,  and  even  stop  one  just  commencing;  but  as  yet  we  cannot 
cure  an  infection  which  is  already  fully  developed,  though  even  here 
there  is  reason  to  believe  that  we  may  possibly  prevent  an  invasion  of 
the  general  system  from  a  diseased  organ,  as  by  the  pneumococcus  from 
an  infected  lung  in  pneumonia.  On  the  whole,  the  serums  which 
simply  inhibit  the  growth  of  bacteria  have  not  given,  as  observed  in 
practice,  conclusive  evidence  of  great  value  in  already  developed  disease. 
This  type  of  serum  loses  much  of  its  bactericidal  properties  quickly 
and  should  not  be  used  when  kept  for  more  than  a  few  weeks. 

Development  of    Antitoxins   together  with   Bactericidal    Substances. — 
Although  the  serum  of  animals  which  have  been  infected  with  any  one 
of  many  varieties  of  bacteria  is  usually  both  antitoxic  and  bactericidal, 
still  one  form  of  these  protective  substances  is  usually  present  almost 
alone;  thus  antitoxic  substances  are  present  almost  exclusively  in  animals 
injected  with  two  species  of  bacteria  which  produce  powerful  specific 
poisons — viz.,  the  bacilli  of  diphtheria  and  tetanus.    When  the  toxins 
of  either  of  these  are  injected  in  small  amounts  the  animals  after  com- 
plete recovery  are  able  to  bear  a  larger  dose  without  deleterious  effects, 
and  these  doses  in  the  more  suitable  animals  can  be  gradually  increased 
until  a  thousand  times  a  previously  fatal   dose  may  be  administered 
without  any  serious  results  whatever.     To  Behring  and  Kitasato  we 
owe  the  discovery  that  this  protecting  substance  accumulates  to  such 
an  extent  in  the  blood  that  very  small  amounts  of  serum  are  sufficient 
to  protect  other  animals  from  the  effects  of  the  true  extracellular  toxins. 
Except  the  diphtheria  and  tetanus  bacilli  a  few  only  of  the  important 
parasitic  bacteria  attacking  man  produce  these  toxins  and  thus  become 
capable  of  causing  the  production  in  the  body  of  antitoxins,  and  even 
these  do  it  to  a  far  less  extent  than  those  of  tetanus  and  diphtheria. 
Following  them  is  the  plague  bacillus,  and  then  the  cholera  spirilla, 
the  typhoid  bacilli,  the  streptococci,  etc.     These  latter  bacteria  when 
injected  excite  more  of  the  substances  which  inhibit  bacterial  growth 
than  of  those  which  neutralize  their  toxins. 

Antitoxin  a  Preventive. — Antitoxin  prevents  the  poisonous  action  of 
toxin.  It  does  not  restore  the  cells  after  they  have  been  injured  by 
the  toxin;  it  is,  therefore,  like  the  bactericidal  substances,  a  preventive 
rather  than  a  cure.  We  find,  experimentally,  that  a  very  much  smaller 
amount  of  antitoxin  will  neutralize  a  fatal  dose  of  toxin  in  an  animal, 
if  given  before  or  at  the  same  time,  than  if  given  only  shortly  after  it. 


ANTAGONISM  B1-T\\  I-l-X  THE  LIVING  BODY  AND  BACTERIA  161 

An  animal  already  profoundly  poisoned  by  the  toxin  is  unaft'ected  by 
any  amount  of  antitoxin. 

Duration  of  Immunity. — The  antitoxins  of  diphtheria  and  tetanus 
are  gradually  eliminated  from  the  body  after  their  injection  or  after 
their  production  from  toxin  injections.  After  the  usual  immunizing 
dose  the  duration  of  immunity  is  only  from  two  to  six  weeks,  the 
period  differing  in  each  individual.  The  elimination  of  the  antitoxin 
takes  place  partly  through  the  urine  and  other  secretions,  and  it  is 
partly  destroyed  in  the  body.  An  animal  which  has  been  highly  im- 
munized will  retain  considerable  amounts  of  antitoxin  for  from  two  to 
four  months. 

Stability  of  Antitoxins. — The  different  antitoxins  vary  as  to  their 
stability  thus:  that  of  diphtheria  is  somewhat  more  stable  than  that 
of  tetanus.  Kept  aseptically  in  cold  and  dark  storage,  and  protected 
from  access  of  air,  the  more  resistant  antitoxins  may  be  preserved 
sometimes  for  a  year  or  two  with  very  little  deterioration  in  strength. 
At  other  times,  however,  from  unknown  causes,  they  are  gradually  de- 
stroyed, so  that  there  may  be  a  loss  of  about  10  per  cent,  per  month. 
A  serum  requires,  therefore,  to  be  tested  every  few  months  if  we  wish 
to  be  assured  of  its  strength  in  antitoxin.  Preservatives,  such  as  car- 
bolic acid,  trikresol,  etc.,  alter  antitoxins  only  very  slightly  when  in 
dilute  solution,  but  in  strong  solution  they  partially  destroy  them.  Heat 
up  to  62°  C.  does  not  injure  them  greatly,  but  higher  temperatures 
alter  them. 

Method  of  Administration. — Antitoxins  and  bactericidal  substances 
are  absorbed  to  a  very  slight  extent  only  when  taken  by  the  mouth — 
certainly  less  than  1  per  cent.  They  must,  therefore,  be  introduced 
subcutaneously  or  intravenously  to  enter  the  body.  The  antitoxic 
serum  does  not  act  against  the  bacteria  directly,  but,  by  neutralizing 
their  poisons,  it  prevents  them  from  acting  as  irritants  to  the  cells, 
and  so  the  soil  for  the  growth  of  the  bacteria  becomes  unsuitable, 
and  they  cease  to  develop. 


1 1 


CHAPTER     XV. 

NATURE   OF  THE   PROTECTIVE   DEFENCES  OF   THE   BODY   AND 

THEIR  MANNER  OF  ACTION— EHRLICH'S 

"SIDE  CHAIN"   THEORY. 

THE  fluids  and  tissues  of  the  animal  body  under  the  normal  condi- 
tions of  life  are,  as  we  have  seen,  not  only  unsuitable  for  the  growth  of 
the  great  majority  of  the  varieties  of  bacteria,  but  even  bactericidal  to 
the  living  organisms  and  antitoxic  to  their  poisons. 

In  seeking  to  account  for  the  bactericidal  property  of  the  blood, 
which  to  a  greater  or  less  extent  affects  all  bacteria,  we  cannot  find  it 
either  in  the  insufficient  or  excessive  concentration  of  the  nutritive  sub- 
stances, nor  in  the  temperature,  nor  in  the  reaction;  for  although  some 
of  these  conditions  may  be  unsuitable  for  some  bacteria,  they  are  all 
suitable. for  many,  and  thus  cannot  constitute  the  fundamental  explana- 
tion of  either  natural  or  acquired  immunity.  We  are  thus  driven  to 
the  conclusion  that  the  body  fluids  and  cells  contain  substances  which 
are  deleterious  to  most  or  all  of  .the  bacteria.  As  to  the  origin  of  these 
substances,  we  may  conceive  that  they  may  be  either  regularly  produced 
by  certain  types  of  the  many  varieties  of  body  cells,  or  that  they  may  only 
be  produced  when  bacteria  or  other  foreign  cells  or  their  substance 
invades  the  body.  When  formed  we  can  conceive  that  they  may  remain 
unaltered  in  the  fluids  for  a  long  period  of  time  or  be  quickly  eliminated 
or  destroyed. 

Bactericidal  Properties  of  the  Blood. — The  bactericidal  effect  upon  most 
bacteria  of  the  blood  serum,  noted  by  Nuttall  in  1888,  is  now  undis- 
puted, and  is  readily  shown  by  the  fact  that  moderate  numbers  of 
bacteria  when  inoculated  into  freshly  drawn  blood  usually  soon  die,  and 
this  destruction  may  be  so  rapid  that  in  a  few  hours  none  of  millions 
remain  alive.  Even  when  some  of  the  bacteria  survive  there  is  for  a 
time  a  decrease  in  the  number  living.  That  this  effect  of  the  blood  is  due 
to  substances  in  the  serum,  and  not  due  to  serum  as  such,  can  be  proven 
by  the  fact  that  not  only  by  injecting  bacteria  into  the  blood  and  peri- 
toneal cavity,  but  also  when  the  bacteria  are  placed  in  the  animal  body 
after  being  enclosed  in  capsules  or  into  serum  contained  in  test-tubes, 
the  bacteria  are  killed  even  if  they  have  previously  grown  outside  the 
body  in  inactive  blood  serum.  Bacteria  have  also  been  injected  into 
a  vein  carefully  ligated  above  and  below,  and  here,  without  coagula- 
tion, the  blood  exerts  bactericidal  properties.  The  germicidal  effect  of 
any  sample  of  blood  serum  on  different  varieties  of  bacteria  is  unequal 
and  can  be  watched  outside  of  the  body.  Here  mixed  with  it  some 
species  of  bacteria  die  quickly,  and  some  lose  only  a  portion  of  their 
number,  those  remaining  alive  after  a  time  rapidly  increasing.  The 


NA  TURE  OF  THE  PROTECTIVE  DEFENCES  OF  THE  BODY   163 

number  of  bacteria  introduced  is  of  great  importance,  for  the  serum 
with  its  contained  substances  is  capable  of   destroying  only  a  certain 
number,  and  after  that  loses  its  bactericidal  properties. 
Thus  the  following  test  was  carried  out : 

Approximate  number  alive  after  being  kept  at  37°  C. 


No.  of  bacteria 

Amount  of 

in  1  c.c.  fluid. 

serum  added. 

One  hour. 

Two  hours. 

Twenty  -seven  hours. 

30,000 

0.1  c.c. 

400 

2 

0 

100,000 

0.1  c.c. 

5,000 

1,000 

200,000 

1,000,000 

0.1  c.c. 

400,000 

2,000,000 

10,000,000 

After  the  proof  given  by  Pasteur  and  his  pupils  as  to  the  existence 
of  acquired  immunity,  attempts  to  explain  it  were  made.  Pasteur  for- 
mulated his  exhaustion  theory,  in  which  an  analogy  was  drawn  between 
an  infection  in  a  living  animal  and  the  exhaustion  of  food  in  a  culture 
media  by  the  growth  of  a  bacterium.  The  knowledge  that  injections 
of  toxins  or  bacterial  protoplasm  was  followed  by  the  production  of 
antibodies  disproved  his  theory  in  the  sense  he  understood  it,  but 
Ehrlich's  theory  of  the  necessity  of  suitable  cell  receptors  to  allow  of 
union  of  poison  to  cell  suggests  the  possibility  that  natural  immunity 
is  sometimes  due  to  the  lack  of  suitable  receptors  or  sensitive  sub-, 
stances. 

Chauveau,  like  Pasteur,  starting  from  facts  observed  in  a  culture, 
considered  that  acquired  immunity  was  due  to  substances  retained 
in  the  body  after  recovery  from  an  infection  which  were  noxious  to 
bacteria.  Here,  again,  later  information  has  changed  the  explanation, 
so  that  we  know  that  it  is  not  substances  left  by  bacteria,  but  delete- 
rious substances  produced  by  the  body  cells  through  the  stimulus  of 
the  bacterial  products. 

During  these  earlier  years  Metchnikoff  perceived  that  the  infected 
host  was  too  little  considered,  and  he  drew  attention  to  the  role  of  the 
leukocytes.  His  original  theory  of  immunity  is  based  on  the  observa- 
tions that  leukocytes  frequently  take  up  bacteria  injected  into  the  blood. 
Metchnikoff  held  that  the  virus  was  destroyed  in  the  interior  of  certain 
mesodermic  cells  by  a  process  of  digestion. 

At  the  same  time  that  this  theory  was  being  developed  another  was 
gradually  being  evolved.  It  was  noticed  that  the  bacteria  injected  into 
the  blood  and  tissues  disappeared.  Nuttall  showed  that  bacteria  are 
destroyed  in  cell-free  serum  but  that  this  property  of  the  serum  is 
destroyed  by  heating  it  at  56°  C.  Buchner  made  many  experiments, 
and  finally  elaborated  his  alexin  theory  of  immunity.  He  showed  that 
bacteria  absorbed  these  bactericidal  substances.  Later,  Bordet,  Ehrlich, 
and  others  established  that  the  alexin  of  Buchner  was  really  a  mixture 
of  two  substances  of  which  one,  named  "  immune  body,"  is  developed 
as  the  result  of  the  injection  of  foreign  cell  substance  and  is  resistant 
to  heat,  and  the  other,  named  "  complement/'  is  present  in  the  blood 
of  normal  animals,  is  not  increased  by  injection,  and  is  unstable. 
Neither  one  of  these  substances  alone  destroy  bacteria,  while  together 
they  do.  Ehrlich  pointed  out  the  similarity  of  complements  to  the  true 
toxins,  such  as  those  produced  by  the  diphtheria  and  tetanus  bacilli. 


PRINCIPLES  OF  BACTERIOLOGY 

During  the  investigations  on  the  bactericidal  substances  of  the  blood 
the  discovery  of  the  antitoxins  was  made  by  Behring  and  Kitasato,  and 
the  nature  of  toxins  was  investigated  by  Roux,  Ehrlich,  and  others. 

Ehrlich's  Theories. — Ehrlich 's  researches  led  to  the  development  of 
his  theories  on  immunity  which  have  had  a  powerful  influence  upon 
all  later  investigations  in  this  field.  His  pupil  and  colleague,  Wasser- 
mann,  explains  them  in  the  following  words: 

Ehrlich  began  by  observing  that  of  the  many  poisonous  substances 
known  to  us  only  a  comparatively  small  number  existed  against  which 
we  could  truly  immunize — i.  e.,  obtain  specific  antibodies  in  the  blood 
serum  of  the  immunized  organism.  Let  us  look  at  two  poisons  which 
are  very  similar  in  their  physiological  action,  for  example,  strychnine 
and  tetanus  poison,  both  of  which  excite  spasms  through  the  central 
nervous  system.  It  is  really  curious  that  the  injection  of  one,  strych- 
nine, produces  no  antibody  whatever  in  the  serum,  while  the  injection 
of  the  other,  the  tetanus  poison,  causes  the  formation  of  the  specific 
tetanus  antitoxin.  Ehrlich  says  that  this  is  because  these  substances 
enter  into  entirely  different  relations  with  the  cells  of  the  living  organ- 
ism. The  one  substance,  strychnine,  merely  enters  into  a  loose  com- 
bination with  the  cells  of  the  central  nervous  system,  so  that  it  can  again 
be  abstracted  from  these  cells  by  all  kinds  of  solvents — e.  g.,  by  shaking 
with  ether  or  chloroform.  The  combination,  therefore,  is  a  kind  of 
solid  solution,  such  as  has  been  shown  for  the  staining  with  aniline 
dyes.  The  tetanus  poison,  on  the  contrary,  Ehrlich  says,  is  firmly 
bound  to  the  cell;  it  enters  the  cell  itself,  becoming  a  chemical  part  of 
the  same,  so  that  it  cannot  again  be  abstracted  from  the  cell  by  solvent 
agents.  He  compares  this  process  to  .the  assimilation  of  nutrient  sub- 
stances. Hence  the  difference  between  these  two  substances  could  be 
likened  to  that  between  saccharin  and  sugar.  Both  substances  taste 
sweet,  but,  despite  this  similarity  in  one  of  their  physiological  actions, 
they  behave  very  differently  toward  the  cells  of  the  organism.  Sac- 
charin simply  passes  through  the  organism  without  entering  into  a 
firm  combination — i.  e.,  without  being  assimilated — and  is  therefore 
no  food.  Its  sweetening  action  is  due  to  a  mere  contact  effect  on  the 
cells  sensitive  to  taste.  Sugar,  on  the  contrary,  is  actually  bound  by 
the  cells,  assimilated  and  burnt,  so  that  it  is  a  true  food.  Ehrlich  says 
that  the  first  requirement  for  every  substance  against  which  we  can 
obtain  a  specific  serum  must  be  its  power  to  enter  into  such  a  combina- 
tion with  certain  particular  cells  in  the  living  organism.  The  substance 
must  possess  a  definite  chemical  affinity  for  certain  parts  of  the  organism. 
Hence,  in  each  substance  against  which  we  can  specifically  immunize, 
Ehrlich  assumes  a  group  of  atoms  which  effects  the  specific  binding 
to  certain  cells,  the  haptophore  group  (Fig.  67,  F).  Corresponding  to  this 
is  a  group  in  the  cell  of  the  living  organism  C,  the  receptor  group,  with 
which  the  haptophore  group  combines.  The  latter  is  entirely  distinct 
from  that  part  of  the  substance  which  exerts  the  physiological  or  path- 
ological effect,  in  toxins,  for  example,  from  the  group  which  is  the  car- 
rier of  the  poisonous  action,  the  so-called  toxophore  group  E,  or  in  fer- 


A.I  TURE  OF  THE  PROTECTIVE  DEFEXCES  OF  THE  BODY   165 

ments,  from  the  group  which  exerts  the  ferment  action,  the  zymophore 
group.  Both  groups,  haptophore  and  functional,  are  independent  of 
each  other,  and  their  separate  presence  can  easily  be  demonstrated 
because  the  functional  group — e.  g.,  in  poisonous  toxins  the  toxophore 
group — is  more  readily  destroyed  by  heat  than  the  haptophore  group. 
Thus  by  heating  a  toxin  for  some  time  to  60°  to  65°  C.  substances 
will  be  obtained  which  are  no  longer  poisonous,  but  which  still  possess 
the  binding,  haptophore,  group.  In  the  case  of  toxins  such  substances 
are  called  toxoids.  Ehrlich  further  stated  that  the  finer  mechanism  of 
the  formation  of  specific  substances  was  this:  that  the  haptophore 
group  was  bound  to  the  receptor  of  the  living  organism  owing  to  a 
specific  affinity.  As  a  result  of  this  the  receptor  is  lost  to  the  living 
organism,  disposed  of,  and  a  biological  law  formulated  by  Weigert 


FIG.  67 


--E 


Graphic  representation  of  receptors  of  the  first  and  third  orders  and  of  complement  as  conceived 
by  Ehrlich:  A,  complement:  B,  intermediary  or  immune  body;  C,  cell  receptor;  D,  part  of  cell: 
E,  toxophorous  group  of  toxin  ;  F,  haptophorous  group. 

now  comes  into  action,  the  law  of  supercompensation;  that  is,  the 
organism  seeks  to  replace  this  defect,  but  in  doing  so  not  merely  replaces 
the  receptors  in  question,  but,  according  to  Weigert,  produces  more 
of* them  than  were  previously  present  (Fig.  68).  The  conditions  are 
somewhat  like  those  seen  in  the  callus  after  a  fracture,  in  which  the 
organism  likewise  does  not  produce  just  the  amount  of  bone  previously 
present;  there  is  always  an  overproduction. 

In  this  way,  Ehrlich  says,  such  a  large  number  of  one  type  of  receptors 
are  produced  by  the  organism  that  these  become  too  much  for  the 
same;  they  are  then  thrust  off  into  the  blood,  and  these  free  receptors 
circulating  in  the  blood  constitute  the  specific  antibodies.  Ehrlich 
therefore  believes  that  the  specific  antibodies  in  the  serum  are  nothing 
else  than  receptors  for  which  the  substance  employed  in  immunization 


166 


PRINCIPLES  OF  BACTERIOLOGY 


possesses  specific  affinity.  Hence,  the  same  substance  which,  so  long 
as  it  remains  in  the  organ,  attracts  the  toxin  and  makes  it  possible  for 
that  to  exert  its  poisonous  action  on  the  organ,  this  same  substance 
acts  as  a  protection  when  it  circulates  outside  of  the  organ;  for  then  it 
satisfies  the  affinity  of  the  poison's  haptophore  group  while  still  in  the 
blood,  preventing  the  poison  molecule  from  acting  on  the  organ  itself. 


FIG.  68 


Graphic  representation  of  Ehrlich's  theory  of  the  production  of  antitoxin  and  the  neutralization 
of  toxin :  a,  diphtheria  toxin  molecule ;  x,  toxophore  atom  group ;  y,  haptophore  or  combining 
group ;  b,  cell  receptors  with  affinity  for  diphtheria  toxin  ;  c,  other  cell  receptors. 

1.  Cell  with  its  receptors.    Outside  of  cell,  free  toxin  molecules. 

2.  Toxin  molecules  combined  with  the  cell  receptors  having  affinity  for  diphtheria  toxin. 

3.  After  three  days,  showing  multiplication  of  cell  receptors  similar  to  those  combined  with  toxin. 

4.  After  four  days,  excess  of  receptors  cast  off  in  the  blood. 

5.  Toxin  molecule  neutralized  by  combining  with  free  receptors  in  blood  of  immunized  animal 
or  in  an  animal  into  which  blood  with  free  receptors  bad  been  transferred. 

In  the  formation  of  the  specific  antibodies  we  must  therefore  dis- 
tinguish three  stages  (Fig.  68) : 

The  binding  of  the  haptophore  group  to  the  receptor  (2) . 

The  increased  production  of  the  receptors  following  this  binding  (3) . 

The  thrusting  off  of  these  increased  receptors  into  the  blood  (4). 

A  considerable  part  of  Ehrlich's  theories  upon  toxins  and  antitoxins 
have  been  confirmed  experimentally;  for  example,  the  presence  of  the 
separate  toxophore  and  haptophore  groups  and  the  existence  of  atom- 
groups  with  specific  binding  properties  in  all  substances  with  which  we 
can  immunize. 


AM  TURK  OF  THE  PROTECTIVE  DEFENCES  OF  THE  BODY  167 

Up  to  the  present  time  the  mechanism  of  the  increased  production  of 
receptors  following  the  .binding  of  the  haptophore  group  to  the  recep- 
tor has  not  been  experimentally  proven.  Wassermann,  however, 
believes  that  he  and  Bruck  have  now  been  successful  in  this.  Although 
I  consider  his  interpretation  as  most  interesting,  yet  both  this  and  the 
reasoning  from  it  are  far  from  proven. 

They  employed  a  tetanus  poison  kept  since  1896.  This  poison  was 
originally  very  toxic.  In  the  course  of  years,  however,  owing  to  the 
damaging  influence  of  this  long  standing,  that  is,  owing  to  the  action 
of  light,  the  oxygen  of  the  air,  etc.,  it  had  become  so  weak  that  it  was 
no  longer  toxic  at  all.  They  were  able  to  give  a  guinea-pig  1  c.c.  with- 
out producing  tetanus.  Nevertheless,  haptophore  groups  had  re- 
mained intact,  as  could  readily  be  proved,  for  this  non-poisonous  tetanus 
toxin  was  still  able  to  bind  tetanus  antitoxin — i.  e.,  thrust-off  receptors. 
When  they  injected  rabbits  with  this  non-poisonous  tetanus  toxoid  in 
increasing  doses,  and  then  examined  the  blood  serum  of  the  animal, 
they  found  not  a  trace  of  tetanus  antitoxin.  They  considered  the  absence 
of  antitoxin  in  this  experiment  could  have  either  of  two  causes : 

1.  It  might  be  that  the  toxoid  no  longer  produced  any  physiological 
effect  whatever  in  the  organism;  or 

2.  Although  it  still  caused  an  increase  in  the  receptors  in  the  cells, 
these  increased  receptors  remained  in  them,  and  were  not  thrust  off 
into  the  blood. 

In  order  to  decide  this  question  they  first  determined  as  nearly  as  pos- 
sible the  exact  quantity  of  fresh  tetanus  toxin  which  constituted  a  fatal 
dose  for  guinea-pigs  of  a  definite  size.  If,  now,  the  action  of  the  tetanus 
toxoid  was  such  that  in  the  living  organism  it  was  not  bound  to  the 
receptors,  it  should  be  possible  to  prove  this  experimentally. 

Their  line  of  reasoning  was  as  follows:  Were  they  to  inject  first  the 
toxoid,  and  shortly  after,  say,  in  one  to  two  hours,  the  fresh  toxin,  they 
would  in  such  an  animal  have  to  increase  the  fatal  dose — i.  e.,  they 
would  require  more  tetanus  toxin  to  kill  this  animal  than  an  untreated 
one,  because  owing  to  the  previous  toxoid  injection  the  most  sus- 
ceptible cell  receptors  had  already  been  occupied.  Provided  Ehrlich's 
theory  was  correct,  so  that  this  binding  of  the  toxoid  really  occurred, 
the  conditions  should  be  entirely  different  when,  instead  of  injecting 
the  toxin  shortly  after  the  toxoid,  they  waited  somewhat  longer,  one 
to  three  days,  and  then  injected  the  fresh  tetanus  toxin.  For  in  that 
case  Weigert's  law  should  come  into  play  and  the  receptors  have  com- 
menced to  increase  in  numbers — i.  e.,  the  organ  would  now  possess 
more  sensitive  groups  than  before.  This  would  have  to  manifest  itself 
in  such  fashion  that  in  contrast  to  the  first  experiment  the  fatal  dose 
of  fresh  tetanus  toxin  could  no\v  be  decreased;  in  other  words,  a  small 
dose  would  now  tetanize  the  animal  in  a  shorter  time. 

As  a  matter  of  fact,  the  experiments  yielded  results  which  were 
exactly  like  those  deduced  theoretically  as  just  described.  They  in- 
jected a  guinea-pig  with  some  of  the  non-poisonous  toxoid,  and  then, 
one  hour  later,  with  the  tetanus  toxin.  They  found  that  much  more 


168  PRINCIPLES  OF  BACTERIOLOGY 

toxin  was  required  to  kill  this  animal  than  a  normal  guinea-pig  of 
equal  size.  If,  on  the  contrary,  they  waited  one  to  three  days,  it  was 
found  that  then  a  dose  of  tetanus  toxin  which  would  not  even  tetanize 
an  untreated  guinea-pig  was  sufficient  to  kill  this  one. 

If  their  explanation  of  the  above  experiments  is  correct,  then  we  have 
for  the  first  time  proof  of  the  existence  of  the  first  two  of  the  three 
stages  demanded  by  Ehrlich's  theory.  The  first  stage,  the  binding 
of  the  haptophore  group,  is  demonstrated  with  the  toxoid;  the  second 
stage,  the  increased  production  of  receptors,  is  demonstrated  by  the 
second  series  of  experiments  just  described ;  the  third  stage  is  the  thrust- 
ing off  of  the  receptors.  Close  attention  to  these  experiments  will  show 
that  with  a  completely  non-poisonous  toxoid  no  receptors  were  thrust 
off  at  all.  The  serum  of  the  rabbit  treated  with  this  toxoid  contained 
no  antitoxin  whatever.  Hence  it  follows  that  the  thrusting  off  of  the 
receptors  into  the  blood  serum  does  not  necessarily  follow  from  their 
overproduction,  but  that  something  additional  is  required.  This  "some- 
thing," which  we  may  term  a  stimulus,  is  a  function  of  the  toxophore 
group.  Because  of  these  experiments  Wassermann  conceives  the 
mechanism  of  the  presence  of  the  specific  antibodies  in  the  serum  to  be 
as  follows:  1.  The  haptophore  group  is  bound  by  the  receptor.  2. 
The  consequence  of  this  binding  is  a  proliferation  of  the  receptors.  In 
this  stage,  however,  they  are  still  attached  to  the  organ.  3.  In  order 
that  they  be  thrust  off,  a  certain  stimulus  is  required.  The  haptophore 
group  is  incapable  of  exerting  this,  so  that,  in  the  case  of  tetanus  toxin, 
this  is  exerted  by  the  toxophore  group.  These  experiments,  although 
important,  do  not  appear  to  me  to  establish  as  much  as  Wassermann 
conceives.  There  is  no  proof  that  additional  receptors  in  the  cells  of 
an  animal  so  sensitive  as  a  guinea-pig  would  in  fact  make  it  respond 
to  a  smaller  dose  of  toxin.  It  would  be  equally  easy  to  argue  that  it 
would  be  less  sensitive,  since  fewer  cells  might  absorb  all  the  toxin. 
The  fact  that  the  cells  combining  with  the  toxoid  yielded  up  no  anti- 
toxin might  be  taken  to  prove  that  the  cells  specifically  attacked  were 
not  the  ones  that  produced  the  antitoxin. 

Other  Explanations  of  the  Production  of  Antitoxins. — Gruber  along  with 
others  consider  that  the  antitoxins  are  not  normal  constituents  of  the 
cells  but  simply  secretions  of  the  cells  under  the  stimulus  of  the  toxin, 
and  the  cells  secreting  are  not  the  cells  sensitive  to  the  toxin,  but 
others — e.  g.,  the  blood-producing  organs.  Gruber  claims  that  the  power 
to  poison  highly  immunized  animals  with  toxin  is  against  Ehrlich's 
ideas.  This  objection  can  be  met  in  several  ways.  Ehrlich  believes 
that  the  receptors  while  still  in  the  cell  may  have  a  greater  affinity 
for  the  toxin  than  after  being  thrown  into  the  circulation.  Gruber 
claims  that  the  latent  period  in  toxin  poisoning  is  due  to  the  slow 
absorption  of  the  toxin  by  the  circulation,  and  does  not  need  the 
elaborate  explanation  of  the  toxophore  group  acting  through  the 
haptophore.  The  idea  that  antitoxin  is  simply  the  toxin  modified  has 
been  given  up  for  one  reason  because  the  antitoxin  developed  is  several 
thousand  times  enough  to  neutralize  the  toxin  injected. 


Y.I  TURE  OF  THE  PROTECTIVE  DEFEXCES  OF  THE  BODY  169 

.  One  objection  against  the  Weigert-Ehrlich  hypothesis  of  overproduc- 
tion of  antitoxin  by  the  specifically  attacked  cells  is  that  while  the 
animals  are  still  showing  tetanic  symptoms  the  receptors  of  the  still 
diseased  cells  are  supposed  to  have  been  reproduced,  as  shown  by  anti- 
toxin production.  This  is  answered  by  Weigert  that  while  the  more 
important  cell  atom  groups  are  still  suffering,  the  groups  producing 
the  receptors  may  have  recovered.  This  supposition  is  difficult  to 
prove  or  disprove. 

The  idea  of  Weigert,  that  the  cells  are  biologically  altered  so  as  to 
continue  to  make  receptors  (antitoxin)  after  the  cessation  of  the  injec- 
tions, is  not  in  accord  with  the  observations  made  by  us,  as  there  is 
uniformly  a  great  drop  in  antitoxin  ten  days  or  two  weeks  after  the 
cessation  of  the  fresh  stimulus  of  renewed  injections.  We  have  also 
shown  that  by  partially  neutralizing  toxin  before  injecting  it  into  ani- 
mals, it  is  possible  to  excite  the  cells  to  produce  as  much  antitoxin  from 
the  first  as  from  any  later  injections. 

We  know  from  the  researches  of  Meyer  and  Ransom  that  tetanus 
poison  is  not  absorbed  by  the  affected  nerve  cells  by  way  of  the  blood 
and  lymph  channels,  but  reaches  them  by  way  of  the  nerves.  Following 
its  injection  the  tetanus  poison  ascends  in  the  axis  cylinder  of  the  nerves 
to  the  motor  cells.  The  tetanus  antitoxin,  unlike  the  toxin,  is  not  a 
neurotrophic  substance,  but  always  follows  the  blood  and  lymph  chan- 
nels. In  adrenalin  we  possess  a  substance  which  is  able  strongly  to 
contract  the  capillaries  and  thus  to  block  the  blood  absorption  path  of 
a  particular  area.  Proceeding  from  these  facts,  Wassermann  and  Bruck 
devised  the  following  experiment:  Tetanus  toxin  and  antitoxin  were 
mixed  in  such  proportions  that  the  mixture  was  entirely  innocuous 
to  animals.  If  this  mixture  was  injected  into  the  hind  paw  of  a 
guinea-pig  no  tetanus  developed.  When,  however,  some  adrenalin  was 
injected  into  the  hind  paw  of  a  similar-sized  guinea-pig,  and  after 
waiting  a  few  minutes  until  the  capillaries  had  contracted,  there  was 
injected  a  recent  mixture  of  tetanus  toxin  and  antitoxin,  typical  tetanus 
was  produced.  WThat  happened  was  this :  that  the  channel  of  absorption 
for  the  tetanus  antitoxin  had  been  blocked,  while  that  for  the  toxin,  the 
nerve  path,  was  open.  The  toxin  had  therefore  torn  loose  from  its 
combination  and  followed  its  course  to  the  central  nervous  system, 
where  it  produced  tetanus. 

This  experiment,  however,  succeeds  only  within  a  certain  period— 
i.  e.,  not  over  an  hour  after  mixing  the  toxin  and  antitoxin.  If  we  wait 
a  longer  time,  say,  three  or  four  hours,  it  will  be  found  that  even  in  the 
adrenalin  animal  no  tetanus  is  produced,  because  by  this  time  the  com- 
bination, previously  a  loose  one,  is  so  firm  that  the  substances  can  no 
longer  be  torn  apart.  This  union  can  be  hastened  by  employing  more 
tetanus  antitoxin,  for  with  an  excess  of  antitoxin  even  after  only  half 
an  hour  it  is  impossible  by  means  of  adrenalin  to  loosen  the  tetanus 
toxin.  This  experiment,  therefore,  shows  that  the  tetanus  toxin-anti- 
toxin combination  is  at  first  a  loose  one,  and  that  the  affinity  becomes 
firmer  and  firmer  with  time.  It  also  shows  the  possibility  of  slightly 


170  PRINCIPLES  OF  BACTERIOLOGY 

increasing  this  affinity  by  means  of  an  excess  of  antitoxin,  a  point  of 
considerable  practical  value  in  serum  therapy. 

Antibacterial  Action.  THE  ACTIVE  SUBSTANCES  IN  BACTERICIDAL 
AND  BACTERIOLYTIC  SERUM. — The  first  important  fact  noted  which 
suggested  that  a  bactericidal  serum  produced  its  action  through  more 
than  one  substance  was  the  discovery  that  while  the  power  of  a  fresh 
bactericidal  serum  to  kill  bacteria  in  the  test-tube  is  lost  by  heating  it  to 
60°  C.,  yet  injected  into  the  peritoneal  cavity  or  blood  of  a  living  animal 
the  power  is  still  exerted.  The  same  is  true  if  in  the  test-tubes  to  the 
heated  serum  there  is  added  some  fresh  normal  serum  which  is  itself 
incapable  of  destroying  the  bacteria.  From  these  observations  the  fact 
became  gradually  apparent  that  the  bactericidal  property  of  a  cell-free 
serum  depended  upon  two  components. 

Different  investigators  have  applied  to  them  different  names.  The 
one  which  is  resistant  to  heat,  which  attaches  itself  directly  to  bacteria, 

FIG.  69 


Graphic  representation  of  amboceptor  or  receptors  of  the  third  order  and  of  complement,  showing 
on  left  the  immune  body  uniting  complement  to  foreign  cell  and  on  right  the  action  of  anticomple- 
ment,  binding  complement:  A,  complement ;  B,  intermediary  body;  C,  receptor;  D,  cell;  E,  anti- 
complement. 

even  at  low  temperatures,  and  is  increased  during  immunization,  is 
called  sensitizing  substance,  interbody,  amboceptor,  or  immune  body. 
The  other,  which  is  sensitive  to  heat,  which  is  present  in  the  healthy 
normal  serum,  is  not  increased  during  immunization,  and  which  unites 
with  the  bacterial  protoplasm  only  at  temperatures  considerably  above 
the  freezing  point,  is  called  alexin,  or  complement. 

The  immune  body  attaches  itself  to  the  bacterial  substance,  but  does 
not  appreciably  harm  the  cells.  The  complement  destroys  the  cell  after 
the  immune  body  has  made  the  cell  vulnerable. 

According  to  Ehrlich  the  immune  body  first  unites  with  the  proto- 
plasm of  the  cell  and  this  develops  in  the  immune  body  an  affinity  for 
the  complement  and  the  two  unite.  (See  Fig.  69.)  He  believes  that 
it  is  through  the  immune  body  that  the  complement  exerts  its  action 
on  the  cell. 

Others  believe  that  the  immune  body  and  complement  do  not  directly 
unite.  It  appears  as  if  the  immune  body  injured  the  cell  membrane 


At  Tl'RE  OF  THE  PROTECTIVE  DEFENCES  OF  THE  BODY     171 

and  so  allowed  the  complement  to  penetrate  the  cell  substance  or 
that,  as  the  French  put  it,  changes  its  nature  so  it  can  combine  with 
its  complement.  Muir  has  shown  that  when  cells  are  saturated  with 
both  immune  body  and  complement  that  the  addition  of  fresh  cells 
causes  a  splitting  oft'  of  immune  body  but  not  of  complement.  This 
throws  further  doubt  upon  the  direct  union  of  immune  body  and  com- 
plement. Most  of  the  experiments  which  have  been  made  with  the 
purpose  of  clearing  up  these  difficult  problems  have  been  made  upon 
red  blood  cells.  Here  the  absorption  of  the  immune  bodies  at  low 
temperatures  and  the  lack  of  noticeable  injury  until  the  complement 
is  added,  at  a  temperature  of  20°  to  30°  C.,  is  very  striking.  The 
immune  bodies  are  very  numerous  and  fairly  specific  in  their  action. 
The  complement  substance  is  much  less  specific  and,  although  probably 
multiple,  each  variety  acts  upon  widely  different  bacteria  after  they 
have  united  with  the  immune  body.  There  is  little  reason  to  think  that 
the  complement  of  one  animal  is  any  more  capable  to  attack  bacteria 
prepared  by  immune  bodies  developed  in  its  blood  than  by  immune 
bodies  developed  in  some  other  species. 

The  building  of  immune  bodies  in  the  infected  animals  is  believed 
by  most  to  take  place  the  more  rapidly  the  more  virulent  the  infecting 
organisms  are.  In  our  experiments  this  has  not  been  evident.  Increase 
of  virulence  for  one  species  of  animal  does  not  mean  increase  for  all 
animals ;  so  that  the  animal  upon  which  the  virulence  is  tested  must  be 
the  same  variety  as  the  one  being  immunized  to  draw  conclusions. 

Origin  of  Bactericidal  Substances. — Ehrlich  and  his  followers  consider 
the  immune  bodies  to  be  built  up  in  the  same  way  as  the  antitoxins. 
They  are  cell  atom-groups,  which  are  similar  to  those  which  were 
attacked  by  the  bacterial  substance  and  which  were  overproduced  as 
the  cell  attempted  to  replace  what  had  been  destroyed  or  bound  up. 

Their  source  must  apparently  be  attributed  to  the  cells,  but  probably 
only  certain  cells  produce  them.  The  red  blood  cells,  for  instance,  seem 
rather  to  destroy  than  to  increase  them.  The  nuclein  derived  from  the 
cells,  although  it  has  a  general  bactericidal  action,  and  may  enter  into 
the  complements  (alexins),  has  different  properties,  and  so  cannot 
itself  be  one  of  these  bodies.  The  cells  which  have  abundant  nuclear 
substance,  such  as  the  leukocytes  and  lymph  cells,  seem  especially  to 
be  a  source,  and  Metchnikoff  asserts  their  pre-eminent  role  as  the  pro- 
ducers of  both  complements  and  immune  bodies.  Buchner  and  others 
have  found  that  through  the  irritation  of  bacterial  filtrates  the  leukocytes 
were  attracted  in  great  numbers  to  the  region  of  injection,  and  that  the 
fluid  here,  which  was  rich  in  leukocytes,  was  more  bactericidal  than 
that  of  the  blood  serum  elsewhere.  Some  claim  to  have  demonstrated 
that  along  with  increased  leukocytosis  there  is  a  general  increase  in  the 
complement  in  the  blood;  still,  it  has  not  yet  been  positively  established 
that  the  complement  is  derived  solely  from  the  leukocytes,  nor  from 
all  leukocytes,  and  a  mere  increase  in  them  does  not  always  mean  an 
increase  in  the  complement.  Immune  bodies  appear  to  be  more  abun- 


172  PRINCIPLES  OF  BACTERIOLOGY 

dant  in  the  spleen  and  the  hsemopoietic  organs  and  also  to  appear  there 
first  during  the  process  of  immunization. 

The  Part  Played  by  the  Leukocyte  in  Immunity . — The  original  theory  of 
Metchnikoff,  that  the  leukocytes  were  the  only  actual  protective  bodies 
which  warded  off  disease,  and  that  they  did  this  by  attacking  the  bacteria 
was  founded  on  the  now  well-known  fact  that  certain  of  the  white  cells 
possessed  the  power  of  taking  up  into  themselves  pathogenic  bacteria, 
and  that  they  were  there  destroyed.  It  was  later  observed  that  these 
cells  had  the  property  of  taking  from  the  blood  many  lifeless  foreign 
elements,  thereby  keeping  the  blood  channels  free  of  foreign  particles. 

The  question  thereby  arose  as  to  whether  these  cells  engulfed  and 
then  killed  the  bacteria,  or  whether  perhaps  other  substances  killed  or 
prepared  them  before  the  cells  took  them  up.  It  is  now  known  that 
the  bacteria  first  unite  with  substances  in  the  serum  and  thus  are  pre- 
pared for  phagocytosis.  This  union  does  not  kill  the  bacteria.  The 
leukocytes  and  the  chemical  substances  of  the  blood  thus  both  play  an 
important  part.  The  death  of  the  bacteria  also  liberates  positive 
chemotactic  substances,  and  the  disintegration  of  the  white  blood  cells 
gives  rise  to  bactericidal  bodies.  We  find  that  phagocytosis  is  most 
marked  when  the  disease  is  on  the  decline  or  the  infection  mild,  but  is 
usually  absent  in  rapidly  increasing  infection.  This  would  seem  to 
indicate  that  the  course  of  the  infection  is  often  already  determined 
before  the  leukocytes  become  massed  at  the  point  of  its  entrance.  The 
first  determining  influence  is  given  by  the  condition  of  the  tissues  and 
the  amount  of  bactericidal  substances  contained  in  them,  and  then, 
later,  in  cases  where  the  bacteria  have  been  checked,  comes  the  addi- 
tional help  of  the  leukocytes.  If  the  tissues  are  wholly  free  of  bacteri- 
cidal and  sensitizing  substances,  neither  they  nor  the  leukocytes,  nor 
both  combined,  can  prevent  the  bacterial  increase.  The  simple  ab- 
sorption by  the  cells  of  bacteria  is  not  necessarily  a  destructive  process. 
Metchnikoff  believes  that  the  polymorphonuclear  leukocytes  are  espe- 
cially antibacterial  in  relation  to  acute  infections.  The  large  phagocytes 
are  conceived  to  deal  chiefly  with  the  resorption  of  tissue  cells  and  with 
immunity  to  certain  chronic  diseases,  such  as  tuberculosis. 

Opsonines1  or  Substances  which  Prepare  the  Bacteria  for  the  Leukocytes. — 
Wright,  Neufeld,  and  others  have  studied  the  substances  in  the  blood 
which  prepare  the  bacteria  for  the  leukocytes.  The  interesting  fact  has 
been  noted  that  the  white  blood  corpuscles  of  the  non-immune  and  the 
actively  immune  have  the  same  phagocytic  characteristics  toward  bac- 
teria, the  differences  in  their  apparent  activity  being  due  to  the  sub- 
stances in  the  serum  which,  similar  to  the  immune  body,  combine  with 
the  receptors  in  the  cells. 

If  the  washed  cells  from  the  immune  patient  are  added  to  salt  solu- 
tion they  have  no  more  effect  upon  the  bacteria  used  to  immunize  than 
do  those  from  a  susceptible  person,  while  those  from  the  latter  put  into 
the  serum  of  the  former  act  energetically.  Heating  destroys  all  opsonines 

1  Greek  "  Opsono  "—I  cater  for. 


NATURE  OF  THE  PROTECTIVE  DEFENCES  OF  THE  BODY     173 

according  to  Wright,  but  some,  only,  according  to  Neufeld.  For  many 
infections,  such  as  those  due  to  the  staphylococci,  streptococci,  and 
piu'umococci,  immunity  seems  to  depend  largely  on  the  opsonines  and 
leukocytes. 

The  opsonic  power  is  measured  by  placing  bacteria  in  a  mixture  of 
the  patient's  serum  and  a  saline  emulsion  of  fresh  white  blood  cells. 
These  are  usually  obtained  from  the  fluid  collecting  after  injecting  an 
aleuronat  emulsion  suspended  in  a  thin  starch  paste  into  the  pleural 
cavity  of  a  rabbit  or  other  animal.  The  fluid  is  withdrawn  twelve  to 
eighteen  hours  after  the  injection.  In  order  to  separate  the  leukocytes 
from  the  pleural  fluid  they  are  washed  in  normal  salt  solution  and 
centrifugalized.  We  have  found  that  suitable  leukocytes  can  be 
obtained  very  readily  from  the  horse.  The  blood  is  received  directly 
into  a  flask  containing  some  10  per  cent,  solution  of  sodium  citrate, 
and  in  such  amount  as  to  make  a  1 :  10  solution.  This  mixture  does 
not  clot.  The  red  cells  sink  to  the  bottom  and  the  supernatant  fluid 
is  centrifugalized. 

After  placing  at  37°  C.  for  from  fifteen  to  sixty  minutes  the  average 
number  of  bacteria  taken  up  by  the  white  corpuscles  is  determined  by 
examining  stained  preparations.  Wright  noticed  that  there  was  no 
opsonic  power  in  the  fluid  in  an  abscess  cavity,  and  that  even  while  the 
blood  as  a  whole  might  have  it,  that  of  some  organ  or  portion  of  the 
body  might  lack  it. 

Deflection  of  the  Complement. — It  frequently  happens  that  when  the 
addition  of  a  small  amount  of  immune  serum  renders  a  normal  serum 
more  bactericidal  a  greater  addition  robs  it  of  all  bactericidal  power. 
This  is  explained  by  Neisser  and  WTechsberg  to  be  due  to  a  locking  up 
of  complement  by  excess  of  immune  body.  The  subject  is  in  need  of 
further  study. 

Multipartial  or  Polyvalent  Sera.  —According  to  Ehrlich's  theory,  every 
immunizing  group  in  a  substance  corresponds  to  a  countergroup  of  the 
fitting  receptors  in  the  organism.  Bacteria  are  not  homogeneous  masses, 
but  are  made  up  of  various  molecules  which  differ  biologically  from 
one  another.  Conforming  to  this,  the  antisubstances,  immune  bodies 
(antitoxins,  agglutinins,  etc.),  which  appear  in  a  serum  are  made  up 
of  the  sum  of  the  antibodies  which  correspond  to  these  partial  ele- 
ments in  the  bacterial  body.  These  separate  groups  are  called  "partial 
groups."  An  immune  serum,  therefore,  consists  of  the  partial  groups 
which  correspond  to  the  separate  partial  elements  of  the  bacterial  body. 
We  are  further  able  to  show  that  these  partial  elements  in  one  and  the 
same  bacterial  species  are  not  the  same  for  all  the  bacteria  of  that 
species.  Thus  one  culture  of  streptococci  or  of  bacillus  coli  may  have 
a  few  partial  elements  which  differ  from  those  of  another  culture. 
What  is  the  consequence  of  this?  The  consequence  will  be  that  when 
we  immunize  with  a  culture  a  of  such  bacteria  we  shall  obtain  a  serum 
which  acts  completely  on  this  culture,  for  in  this  serum  all  the  partial 
elements  present  in  culture  a  are  represented.  If,  however,  AVC  employ 
culture  b,  c,  or  <7,  which  perhaps  possess  other  partial  elements,  we 


174  PRINCIPLES  OF  BACTERIOLOGY 

shall  find  that  the  serum  does  not  completely  affect  these  cultures. 
As  already  stated,  such  a  condition  of  things  is  met  with  in  inflammations 
due  to  streptococci  and  other  bacteria,  and  is,  therefore,  of  considerable 
practical  importance.  It  is  because  of  this  fact  that  a  serum  acts  best 
only  in  a  certain  percentage  of  cases.  In  order  to  overcome  this  difficulty 
in  persons  infected  with  these  bacterial  species  we  have  no  choice  but  to 
make  sera,  not  by  means  of  one  culture,  but  by  means  of  a  number  of 
different  strains  of  the  same  species.  The  result  of  this  will  be  that, 
corresponding  to  the  various  partial  elements  in  these  different  cuL- 
tures,  we  shall  obtain  a  serum  containing  a  large  number  of  the  partial 
groups.  Such  a  serum  will  then  exert  a  specific  action  on  a  large  num- 
ber of  different  cultures,  but  not  quite  as  great  an  influence  on  any- 
one as  if  only  that  variety  had  been  injected. 

In  other  words,  the  development  and  the  closer  analysis  of  the  prob- 
lem of  immunity,  especially  during  the  past  few  years,  have  shown  us 
that  we  must  make  use,  more  than  heretofore,  of  so-called  polyvalent 
or  multipartial  sera.  In  the  serum  therapy  of  streptococcus  infections,  of 
dysentery,  etc. ,  the  production  of  such  multipartial  sera  is  an  advantage 
in  practice.  Owing  to  these  partial  groups  also,  a  serum — e.  g.,  anti- 
typhoid serum — can  specifically  affect  a  closely  allied  species  of  bac- 
*  terium,  like  bacillus  coli,  for  example.  For  it  is  known  that  closely 
related  species  of  bacteria,  such  as  typhoid  and  colon  bacilli,  possess 
certain  partial  groups  in  common,  and  a  serum  is  thus  produced  which 
to  a  certain  extent  acts  on  both  species.  This  constitutes  what  is 
known  as  the  "group  reaction." 


CHAPTER    XVI. 

THE  NATURE  OF  THE  SUBSTANCES  CONCERNED  IN 
AGGLUTINATION. 

THE  agglutinating  substances,  which  develop  in  animals  because  of 
bacterial  infection,  have  proven  of  such  value  in  the  identifying  of 
bacteria  and  the  detection  of  bacterial  infection  that  a  knowledge  of 
them  is  of  great  practical  as  well  as  theoretical  importance.  (See  pages 
81-83  for  technique  of  investigation.) 

The  agglutinins  were  discovered  by  Gruber  and  Durham.  Their 
effect  on  bacteria  can  be  observed  either  macroscopically  or  microscopi- 
cally. For  example,  if  a  serum  from  an  animal  which  has  passed 
through  a  typhoid  infection  is  added  to  a  twenty-four-hour  culture  of 
typhoid  bacilli,  and  the  mixture  placed  in  a  thermostat,  the  following 
phenomenon  will  be  noticed:  The  bacteria,  which  previously  clouded 
the  bouillon  uniformly,  clump  together  into  little  masses,  settle  to  the 
sides  of  the  test-tube,  and  gradually  fall  to  the  bottom  until  the  fluid 
is  almost  entirely  clear.  In  a  control  test,  on  the  contrary,  to  which 
no  active  serum  is  added  the  fluid  remains  uniformly  cloudy.  The 
reaction  is  completed  in  from  one  to  twelve  hours.  If  the  reaction  is 
observed  in  a  hanging  drop,  it  is  seen  that  the  addition  of  the  active 
serum  first  produces  an  increased  motility  of  the  bacteria  which  lasts 
a  short  time  and  is  followed  by  a  gradual  formation  of  clumps.  Fre- 
quently one  sees  bacteria  which  have  recently  joined  a  group  make 
violent  motions  as  though  they  were  attempting  to  tear  themselves  away; 
then  they  gradually  lose  their  motility  completely.  Even  the  larger 
groups  of  bacteria  may  exhibit  movement  as  a  whole.  After  not  more 
than  one  or  two  hours  the  reaction  is  completed ;  in  place  of  the  bacteria 
moving  quickly  across  the  field,  one  sees  one  or  several  groups  of  abso- 
lutely immobile  bacilli.  Now  and  then  in  a  number  of  preparations  one 
sees  a  few  separate  bacteria  still  moving  about  among  the  groups.  If  the 
reaction  is  feeble,  either  because  the  immune  serum  has  been  highly 
diluted  or  because  it  contains  very  little  agglutinin,  the  groups  are  small 
and  one  finds  comparatively  many  isolated  and  perhaps  also  moving 
bacteria.  It  is  essential  each  time  to  make  a  control  test  of  the  same 
bacterial  culture  without  the  addition  of  serum.  Under  some  circu in- 
stances the  reaction  proceeds  with  extraordinary  rapidity,  so  that  the 
bacilli  are  clumped  almost  immediately.  By  the  time  the  microscopic 
slide  has  been  prepared  and  brought  into  view,  nothing  is  to  be  seen 
of  any  moving  or  isolated  bacteria,  and  only  by  means  of  the  control 
test  is  it  possible  to  tell  whether  the  culture  possessed  normal  motility. 
As  to  the  nature  of  these  phenomena  a  number  of  theories  have  been 


176  PRINCIPLES  OF  BACTERIOLOGY 

advanced.  As  in  the  case  of  the  immune  body  there  is  positive  proof 
that  the  agglutinin  combines  directly  with  substances  in  the  bacterial 
body. 

In  some  cases  the  agglutinins  are  active  even  in  very  high  dilutions. 
Thus  in  typhoid  patients  and  typhoid  convalescents  a  distinct  agglu- 
tination has  been  observed  in  dilutions  of  1:5000,  and  this  action 
persisted  for  months,  though  not,  of  course,  in  the  same  degree.  Even 
normal  blood  serurn,  when  undiluted,  often  produces  agglutination. 
But  the  specific  agglutinins,  which  are  formed  only  in  consequence  of 
an  infection,  are  characterized  by  this,  that  they  produce  agglutination 
even  when  the  serum  is  highly  diluted,  and,  furthermore,  that  after  this 
dilution  the  action  is  specific — i.  e.,  the  dilution  of  cholera  immune 
serum  agglutinates  only  cholera  bacilli,  typhoid  immune  serum  only 
typhoid  bacilli,  etc.  This  specificity,  however,  as  will  be  shown  later, 
is  not  always  absolute. 

The  agglutinating  substances  when  mixed  with  bacteria  are  bound 
to  the  agglutinable  substances  in  them,  the  two  bodies  effecting  a  loose 
combination  very  like  toxin  and  antitoxin.  By  chemical  means  it  is 
possible  to  again  separate  the  agglutinin  from  the  bacteria  and  use  it 
to  agglutinate  bacteria  anew. 

It  was  formerly  assumed  that  agglutination  was  a  prerequisite  for 
bacteriolysis.  This,  however,  is  not  so,  for  both  in  cholera  and  in  typhoid 
immunity  bacteriolytic  substances  have  been  observed  without  agglu- 
tinins, and  agglutinating  substances  without  bacteriolysins. 

Of  the  three  antibodies  mentioned,  serum  therapy  has  thus  far  made 
use  of  the  antitoxins;  whereas,  in  serum  diagnosis  the  bacteriolysins 
and  above  all  the  agglutinins  are  used.  Serum  diagnosis  by  means  of 
these  two  substances  was  possible  only  because  they  had  proven  them- 
selves in  general  as  specific. 

Precipitins. — If  we  inject  an  animal  with  albuminous  bodies  of  the 
greatest  variety,  substances  will  usually  appear  in  the  blood  which  pos- 
sess distinct  relations  to  these  bodies.  They  manifest  themselves  by 
their  power  to  precipitate  the  albuminous  bodies  from  dilute  solutions 
in  a  test-tube.  These  antibodies  are  therefore  called  precipitins. 
A  phenomenon  discovered  by  R.  Kraus  is  probably  to  be  separated 
from  this  precipitin  action  on  albumins.  This  author  showed  that  the 
serum  of  a  rabbit  immunized  against  typhoid  produces  a  precipitate  in 
the  bacterial-free  filtrate  of  a  bouillon  typhoid  culture.  This  fact  has 
been  verified  by  a  number  of  investigators  and  found  to  extend  also  to 
other  species  of  bacteria.  The  precipitins  are  divided  like  agglutinins 
into  having  group  and  specific  action. 

Characteristics  of  Agglutinins. — As  considered  by  Ehrlich's  school,  the 
agglutinin  consists  of  a  stable-combining  group  and  an  unstable-precipi- 
tating group.  The  agglutinable  molecule  is  also  believed  to  consist  of 
two  groups,  one  stable  that  combines  with  the  agglutinins  and  one  labile 
that  gives  the  completed  reaction.  Agglutinins  changed  by  heat,  acids, 
and  other  influences  become  agglutinoids,  which  are  comparable  to 
toxoids,  complementoids,  etc. 


\ATURE  OF  SUBSTANCES  CONCERNED  7.V  .{GGLUTL\ATH>\     177 

The  union  of  agglutinin  with  receptors  in  bacteria  is  a  chemical  reac- 
tion, and  is  quantitative.  Before  agglutination  occurs  sodium  chloride 
must  be  present  as  it  enters  into  the  combination.  The  amount  of  bac- 
teria in  the  emulsion  used  to  test  the  amount  of  agglutinin  must,  there- 
fore, be  known.  An  emulsion  one  hundred  times  as  dense  as  another 
would  require  one  hundred  times  as  much  agglutinin  to  give  an  equally 
complete  reaction.  Agglutinin  acts  upon  dead  bacteria. 

Many  things  affect  the  agglutinable  substance  in  bacteria.  Grown 
in  bouillon  for  three  days  at  37°  C.,  bacilli  requires  three  times  as  much 
agglutinin  to  give  an  equal  reaction  as  if  grown  at  37°  C.  for  one  day. 
This  difference  is  partly  due  to  bacterial  substance  passing  out  into 
the  culture  medium,  which  combines  with  the  agglutinin. 

Heat  diminishes  the  agglutinability  of  bacteria  when  above  60°  C. 
Dreyer  found  that  if  a  twenty-four-hour  bouillon  culture  of  bacillus 
coli  required  1  part  of  agglutinin  to  agglutinate  it,  then  if  heated  to 
60°  C.  it  required  2.3  parts;  if  to  80°  C.,  18  parts;  if  to  100°  C.,  24.6 
parts.  He  found  the  surprising  fact  that  long  heating  of  the  culture 
restored  its  ability  to  be  agglutinated  by  smaller  amounts  of  agglutinins. 

Heated  thirteen  hours  to  100°  C.,  the  culture  was  agglutinated  by 
4  parts.  Dreyer's  explanation  of  this  result  is  that  agglutinin-fixing 
substance  is  dissolved  out  by  the  prolonged  heating. 

Heating  the  serum  above  60°  C.  injures  the  agglutinin  slightly,  above 
70  C.  greatly,  and  above  75°  C.  destroys  it.  Weak  and  strong  acids 
agglutinate  bacteria  while  medium  acidity  does  not.  Alkalies  inhibit 
agglutination. 

The  nature  of  agglutinoids  and  the  means  by  which  they  inhibit  agglu- 
tination is  at  present  little  understood.  It  is  important  to  remember  that 
in  concentrated  serum  agglutination  may  fail  because  of  their  action, while 
in  higher  dilutions  of  the  serum  agglutination  may  take  place  readily. 

The  growth  of  bacteria  in  fresh  blood  or  its  equivalent  inhibits  the 
development  of  agglutinable  substance  in  bacteria.  Bacteria  should 
not  be  grown  on  such  media  when  they  are  to  be  used  in  agglutination 
tests.  Even  ascitic  fluid  broth  has  some  effect. 

Group  Agglutination. — Many  varieties  of  bacteria  have  among  the 
different  substances  composing  their  bodies  some  of  which  are  common 
to  other  bacteria  which  are  more  or  less  allied  to  them.  These  sub- 
stances all  exciting  agglutinins  we  have  such  a  serum  acting  on  a 
number  of  varieties.  These  agglutinins  are  called,  therefore,  group 
agglutinins.  If  a  typhoid  or  paratyphoid  serum  possess  a  high  degree 
of  activity — i.  e.,  ability  to  agglutinate  even  in  large  dilution — it  may 
happen  that  with  lesser  dilution  it  may  also  agglutinate  the  two  related 
bacilli.  Thus,  in  two  cases  the  infecting  paratyphoid  bacilli  type  B 
were  agglutinated  1:5700;  typhoid  bacilli,  however,  only  1:120,  while 
paratyphoid  bacilli  type  A  were  not  agglutinated  at  all.  In  two  other 
cases  I  observed  an  agglutination  of  paratyphoid  type  B  with  a  dilution 
1:40,  while  typhoid  bacilli  were  agglutinated  with  1:300  and  over. 
Korte  has  frequently  observed  that  typhoid  sera  agglutinate  not  only 
typhoid  bacilli,  but  also  one  or  both  varieties  of  paratyphoid,  even 

12 


178  PRINCIPLES  OF  BACTERIOLOGY 

when  a  simultaneous  infection  with  paratyphoid  was  excluded.  This 
agglutination  for  the  other  bacilli  was  in  some  cases  quite  marked, 
though  there  was  no  uniformity  whatever.  Since  he  found  that,  con- 
versely, in  paratyphoid  infection  the  serum  possesses  a  fairly  strong 
agglutinating  action  on  typhoid  bacilli,  Korte  advises  that  in  every  case 
of  typhoid  all  three  bacteria  be  tested  for  agglutination,  so  that,  accord- 
ing to  the  strongest  agglutinating  action,  one  can  decide  which  infection 
is  present.  If  in  practice  it  is  immaterial  whether  this  point  be  decided, 
the  agglutination  with  paratyphoid  need  only  be  undertaken  when  the 
typhoid  agglutination  is  absent. 

In  all  this  we  are  dealing  with  the  same  phenomenon  which  undoubt- 
edly plays  a  role  in  the  agglutination  with  blood  of  icteric  patients,  the 
so-called  group  agglutination,  as  it  was  first  termed  by  Meinhard  Pf  aund- 
ler.1  In  other  words,  while  agglutinins  may  be  nearly,  if  not  quite, 
specific  in  their  action  a  serum  which  produces  agglutination  may  be 
far  from  being  so.  As  a  rule,  the  agglutination  with  the  infecting  agent 
is  by  far  the  strongest — i.  e.,  it  proceeds  even  in  high  dilutions — whereas 
other  bacteria  require  a  stronger  concentration.  The  bacteria  which 
are  agglutinated  by  one  and  the  same  serum  need  not  at  all  be  related  in 
their  morphological  or  other  biological  characteristics,  as  at  first  assumed. 
Conversely,  micro-organisms  which,  because  of  the  characteristics  men- 
tioned, are  regarded  as  entirely  identical  or  almost  so,  are  sharply 
differentiated  by  means  of  their  agglutination.  In  other  words,  the 
"groups"  arrived  at  by  means  of  a  common  agglutination  have  no 
relation  to  species  as  the  term  is  usually  employed.  Thus,  according 
to  Stern,  certain  varieties  of  proteus  and  of  staphylococci  excite  the 
production  of  sera  which  exert  marked  agglutinating  powers  also  on 
typhoid  bacilli,  although  otherwise  we  do  not  regard  these  three  micro- 
organisms as  at  all  related.  Because  of  this  lack  of  absolute  specificity 
the  serum  diagnosis  of  infection  or  identification  of  bacteria  has  value  only 
when  very  carefully  tested. 

The  Relative  Development  of  Specific  and  Group  Agglutinins. — The 
study  of  a  large  number  of  series  of  agglutination  tests  obtained  from 
young  goats  and  rabbits  injected  chiefly  with  typhoid,  dysentery,  para- 
dysentery,  paracolon,  colon,  and  hog-cholera  cultures  has  shown  that 
there  is  considerable  uniformity  in  the  development  of  the  specific  and 
group  agglutinins.  The  specific  agglutinins  develop  in  larger  amount 
in  the  beginning,  being  in  the  second  week  usually  from  five  to  one 
hundred  times  as  abundant  as  the  group  agglutinins.  Later  the  total 
amount  of  the  group  agglutinins  tends  to  approach  more  nearly  to 
that  of  the  specific,  and  reach  as  high  as  50  per  cent.  In  a  number  of 
tests  carried  out  by  us  we  found  that  many  group  agglutinins  supple- 
ment specific  ones  in  their  action,  causing  by  their  addition  an  increased 
agglutinating  strength.  In  our  experience  the  variety  of  micro-organ- 
ism used  for  inoculation  is,  if  equally  sensitive,  agglutinated  by  the  com- 
bined specific  and  group  agglutinins  produced  through  its  stimulus 

1  Ueber  Gruppenagglutination  und  das  Verhalten  des  Bacterium  coli  bei  Typhus,  Munch,  med. 
Wochenschrift,  1899,  No.  15. 


A  1  TURE  OF  SUBSTANCES  CONCERNED  IN  AGGLUTINATION    179 

in  a  higher  dilution  than  any  micro-organism  affected  merely  by  the 
group  agglutinins.  It  is  true  that  bacteria  not  injected  were  at  times 
agglutinated  in  higher  dilutions  than  the  variety  injected;  this,  if  not 
due  to  greater  sensitiveness,  was  on  account  of  normal  group  agglu- 
tinins present  in  the  animal  before  immunization.  In  horses  and  adult 
goats  it  was  found  that  before  injections  were  commenced  there  was 
often  a  great  accumulation  of  agglutinins  for  bacteria  and  especially 
for  members  of  the  dysentery,  paradysentery,  and  colon  groups,  so 
that  the  estimation  of  the  development  of  specific  agglutinins  was  a 
matter  of  great  difficulty  except  through  careful  absorption  experiments. 
For  this  reason  untreated  horse  serum  is  a  very  dangerous  substance 
to  use  in  differentiating  the  intestinal  bacteria.  This  is  clearly  brought 
out  in  the  record  given  of  the  tests  made  of  two  horses.  The  great 
height  to  which  the  group  agglutinins  may  rise  is  seen  in  the  following 
table: 

TABLE  I. 

Agglutinin  in  the  Serum  of  a  Horse  Injected  with  Paradysentery  Bacillus. 
Type,  Manila  Culture. 

After  18  injections.  After  21  injections. 

Culture.  1:3000    1:5000    1:10,000    1:3000    1:5000    1:10,000 

Paradysentery  type,  Manila  4-4-  4-4-  4-4- 

Colon  B.  X 4-4-        4-+  4-4-          4-4- 

The  great  amount  of  agglutinins  acting  upon  the  colon  bacillus  X. 
is  remarkable.  A  serum  is  here  seen  to  be  acting  in  dilutions  as  high 
as  1 : 10,000  upon  a  culture  possessing  very  different  characteristics 
from  the  one  used  in  the  injections. 

Although  a  considerable  proportion  of  the  group  agglutinins  acting 
on  colon  bacillus  X  was  undoubtedly  due  to  the  stimulus  of  the  injections 
of  the  Flexner  paradysentery  culture,  still  a  portion  of  them  was  prob- 
ably due  to  other  causes.  In  Table  II.  is  seen  the  marked  accumula- 
tion of  agglutinins  which  may  occur  in  a  horse  before  injections  are 
begun  and  the  results  of  injection  nutrient  bouillon  which  had  been 
prepared  from  meat  in  the  usual  way. 

TABLE  II. 

A  young  horse  before  The  same  one  week  after  being 

inoculation.  injected  with  one  litre  of  bouillon. 


Culture.  1:100  1:500    1:1000    1:5000     1:100      1:500      1:1000    1:5000 

Dysentery  B.,  Japan .       .     4-  4-+         — 

Paradysentery,  Mt.  Desert  4-4- 

Manila     .  4-4- 

ColonB.  X.        ...  4-4-4-  + 

The  fact  of  most  importance  which  appears  in  this  table  is  the  abun- 
dant agglutinins  which  may  be  found  in  the  serum  of  a  horse  which 
has  never  received  bacterial  injections. 

Three  rabbits  injected  with  nutrient  bouillon  developed  agglutinins 
for  the  paradysentery  and  some  of  the  colon  bacilli.  In  one  rabbit's 
serum  the  Manila  culture  agglutinated  in  dilutions  of  1 : 150. 


180 


PRINCIPLES  OF  BACTERIOLOGY 


TABLE  III. 

Serum  from  Two  Horses  at  the  End  of  One  Year,  during  which  Weekly  Injections  were  Given. 

Dysentery  B.  (Shiga).        Paradysentery  (Manila). 

Culture.  l':  500    1  : 1000    1 :  5000        iTsOO    1  :  1000    1 :  5000 

Dysentery  B.,  Japan  +  +  + 

Paradysentery  B.,  Mt.  Desert  +  +  + 

"      Manila.       ..+++  +  -t-  + 

It  is  interesting  to  note  that  the  serum  from  the  horse  receiving  the 
Shiga  culture  agglutinates  the  paradysentery  culture  in  higher  dilutions 
.than  the  Shiga  culture.  In  the  other  serum  all  cultures  were  agglu- 
tinated in  equally  high  dilutions  in  spite  of  the  fact  that  the  paradysen- 
tery type  had  been  injected. 

THE  RELATIVE  ACCUMULATION  OF  THE  GROUP  AND  SPECIFIC  AGGLU- 
TININS.— This  is  seen  to  vary  for  the  different  types  and  at  different 
times.  For  the  Manila  culture  of  Flexner,  which  is  nearest  to  the  colon 
in  its  characteristics,  the  specific  agglutinins  were  in  the  serum  of  an 
animal  which  had  received  injections  of  the  Manila  cultures  at  the 
end  of  the  third  month  six  times  as  abundant  as  the  group  agglu- 
tinin  acting  on  the  Maine  culture  of  Park,  which  represents  a  type 
farther  removed  from  the  colon.  At  the  end  of  the  fourth  month  they 
were  fourteen  times  as  abundant.  For  the  dysentery  bacillus  (Shiga) 
the  development  of  agglutinins  was  the  least. 

Another  point  of  interest  is  that  the  proportional  amount  of  agglu- 
tinins from  the  different  cultures  varied  at  different  times.  If  on  tests 
made  of  a  single  bleeding  we  had  attempted  to  draw  conclusions  as  to 
the  relative  development  of  specific  and  group  agglutinins  between 
the  cultures,  we  would  have  had  an  imperfect  view.  Many  conflicting 
statements  in  literature  are  undoubtedly  due  to  this  lack  of  apprecia- 
tion of  the  variability  in  the  relative  amount  of  these  two  types  of  agglu- 
tinins during  a  long  process  of  immunization. 

The  development  of  group  agglutinins  for  the  three  dysentery  types 
caused  by  the  injections  of  a  colon  bacillus : 


1st 


2d 


FIG.  70 

3d  4th 


5th 


6th* 


7th 


1     500 

m  

—  —  ^ 

1     400 

~? 

\ 

1     300 

/ 

/ 

\ 

1     200 

X 

/ 

x\ 

v 

1      100 

/ 

/' 

x               \ 

\ 

1        00 

—  «^s^ 

—  - 

— 

rrr>a. 

s-^X 

The  rise  and  fall  of  common  and  specific  agglutinins  during  seven  months  in  a  rabbit 

injected  with  the  Manila  culture  after  it  had  been  heated  to  115°  C. 
—  Colon  bacillus  X. 


—  Paradysentery  type  (Manila). 

—  Paradysentery  type  (Mt.  Desert).       Dysentery  type  (Japan). 

Test  dates  for  all  four  sera. 

*  Injections  stopped. 


\.\TURE  OF  SUBSTA\r/;s  CONCERNED  L\T  AGGLl'TI  \  \TIOX    181 


Similar  conditions  to  those  noted  in  previous  chart,  except  that  a  young  goat  has  been  used  for  the 
injections  of  the  colon  bacillus  X.  The  great  accumulation  of  common  agglutinins  for  the  para- 
dysentery  bacillus  in  the  third  month  of  the  injections  of  the  bacillus  X  is  very  striking. 

•  Tests  made. 

The  Use  of  Absorption  Methods  for  Differentiation  between  Specific 
and  Group  Agglutinins  due  to  Mixed  Infection  and  to  a  Single  Infection. — 
It  is  now  well  established  that  if  an  infection  is  due  to  one  micro-organ- 
ism there  will  be  specific  agglutinins  for  that  organism  and  group- 
agglutinins  for  that  and  other  more  or  less  allied  organisms.  If  infec- 
tion is  due  to  two  or  more  varieties  of  bacteria,  there  will  be  specific 
agglutinins  for  each  of  the  micro-organisms  and  group  agglutinins 
produced  because  of  each  of  them. 

The  above  facts  have  been  demonstrated  by  several  investigators. 
The  following  experiments  selected  from  those  reported  by  Castellani1 
well  illustrate  these  points:  A  rabbit  immunized  to  B.  typhi  agglu- 
tinated B.  typhi  1:5000,  B.  coli  (31)  1:600.  After  saturation  with  B. 
typhi  all  agglutinins  were  removed  for  both  micro-organisms.  A  rabbit 
immunized  to  both  B.  typhi  and  B.  coli  (31)  agglutinated  B.  typhi 
1 : 4000,  B.  coli  (31)  1 : 1000.  (After  saturation  with  B.  typhi  the  serum 
did  not  agglutinate  B.  typhi,  but  B.  coli  (31)  1:900.)  After  saturation 
with  B.  coli  it  failed  to  agglutinate  B.  coli  (31),  but  still  agglutinated 
B.  typhi  1:4000. 

From  these  and  other  experiments  Castellani  drew  the  important 
conclusions : 

1.  The  serum  of  an  animal  immunized  against  a  certain  micro- 
organism, when  saturated   with  that  micro-organism,  loses  not  only 
its  agglutinating  power  for  that  organism,  but  also  for  all  other  varieties 
that  it  formerly  acted  upon.    Saturated  with  the  others,  its  action  upon 
the  first  is  reduced  little  or  not  at  all. 

2.  The  serum  of  an  animal  immunized  against  two  micro-organisms, 
A  and  B,  loses  its  agglutination  when  saturated  with  A  only  for  A. 
Saturated  with  A  and  B  it  loses  its  agglutinating  power  for  both. 

3.  These  facts  may  be  applied  to  the  diagnosis  of  an  unknown  mixed 

i  Zeitschrift  f.  Hyg.,  Bd.  atl.,  8. 17. 


182 


PRINCIPLES  OF  BACTERIOLOGY 


infection.  Suppose,  for  instance,  the  serum  from  a  typhoid  case  agglu- 
tinates both  the  laboratory  cultures  of  the  typhoid  bacilli  and  those 
of  a  variety  of  the  colon  group.  We  saturate  the  serum  with  typhoid 
bacilli.  If  the  serum  loses  its  agglutinating  power  for  the  typhoid 
bacillus  only,  it  is  a  case  of  mixed  infection  with  both  the  typhoid 
bacillus  and  the  type  of  colon  bacillus  used  in  the  test.  If  the 
serum  loses  its  agglutination  for  both  the  B.  typhi  and  the  B.  coli, 
then  it  is  a  pure  typhoid  infection,  the  B.  coli  having  been  agglu- 
tinated by  the  group-agglutinins  produced  because  of  the  typhoid 
infection. 

The  conclusions  Castellani  derived  from  the  facts  stated  in  para- 
graphs 1  and  2  are  not  warranted,  because  of  the  fact  that  bacteria 
absorb  group  agglutinins  produced  by  other  varieties  of  bacteria  and 
which  agglutinins  may  not  appreciably  affect  them.  The  agglutinins 
in  the  serum  of  the  supposed  case  of  typhoid  fever  which  agglutinated 
the  test  culture  of  B.  coli  and  were  absorbed  by  B.  typhi  were  not,  it 
is  true,  produced  by  the  variety  of  B.  coli  of  the  test  culture,  but  they 
may  have  been  produced,  and  in  fact  probably  were,  by  some  other 
variety  of  B.  coli.  The  B.  typhi  is  less  apt  to  produce  abundant  group 
agglutinins  for  B.  coli  than  are  other  varieties  of  B.  coli,  and  it  absorbs 
the  group  agglutinins  produced  by  many  varieties  of  the  B.  coli  for 
other  bacteria. 

The  results  of  a  number  of  experiments  carried  out  by  us  demon- 
strate this.  The  following  tables  give  the  outcome  of  several  experi- 
ments : 

ABSORPTION  BY  THE  TYPHOID  BACILLUS  OF  GROUP  AGGLUTININS  ACTING  UPON  A 

NUMBER  OF  VARIETIES  OF  B.  COLI  WHICH  WERE  PRODUCED  BY 

ANOTHER  VARIETY  OF  B.  COLI. 

Agglutination  by  Serum  of  Rabbit  Immunized  to  Colon  Bacillus  X. 

After  attempt  at  absorption 
with  typhoid  bacilli  at  22°  C. 
600 
20 
80 
30 
10 

less  than    10 
<.      .<       10 

10 

The  absorption  tests  were  carried  out  by  adding  the  bacilli  from 
recent  agar  cultures  to  a  10  per  cent,  solution  of  the  serum  in  a  twenty- 
four-hour  bouillon  culture.  The  mixture  was  allowed  to  stand  for 
twenty-four  hours  at  about  22°  C.  It  was  found  that  a  simple  dilution 
of  serum  when  left  at  37°  C.  rapidly  deteriorated.  Thus,  in  an  extreme 
instance  a  serum  positive  at  1 : 1500,  when  diluted  with  bouillon  or 
salt  solution  1 : 25  and  left  at  37°  C.  for  twenty-four  hours,  lost  30  to 
40  per  cent,  of  its  strength;  at  22°  C.  it  lost  15  to  20  per  cent.  Left  for 
three  hours  only,  the  loss  was  only  5  to  10  per  cent. 


>lon  baci 
fphoid 

llusX    .... 
1     .... 

Before  addition  of 
typhoid  bacilli. 
.    600 
.    500 

2      .... 
3     .... 
4     .... 
5     .... 

6-18 

.    500 
.    250 
.    250 
.      10 
less  than      10 
«       «       10 

XATURE  OF  SUBSTANCES  CONCERNED  IX  AGGLUTINATIOX     183 

FIG.  72 


SOOOu,                                                                                            ^ 

rH  •*                                                                                                                                                                                             .**                                                                            ~        *4 

MI800 

6                                      o                                        3    *>                            .0    ^ 
=                                     S    «                             u    '     i;                       L-S2 

1:1600 

o>                                                                            a»    .2     in                        o    ™  </) 
1/1                                       |   £                             £     S    «                        £   J  -c 

1:1400 

•£                                          *    *                                     1    Z                     Manila*    * 

1:1200 

o                                     **                          Manila 

t 

IMOOO 

Httem.     Coney                        t 

01 

• 
«> 

I:  800 

Si  '^         ^  s 

1 

I:  600 

1                      i  s. 

ti      ~> 

§           -o 

1    -° 

I:  400 

j      | 
1        '                                    1              « 

4  t« 

\    %'  - 
\    * 

I:  200 

i   1               i   i  *1 

in 

u 

i:   100 

*     '               o  £ 

1                      P  1     :    °* 

0    0     0 

«->       z 

1  -%£ 

I:     00 

T  1  1    I    !  ! 

£    1     ' 

1     1     ! 

f     !  i 

Showing  the  effect  of  saturating  with  bacilli  of  types  Shiga-Manila  and  Mt.  Desert,  a  serum  from 
a  horse  which  had  received  combined  injections  of  dysentery  bacilli  of  the  three  types.  Note  that 
the  Manila  type  removed  almost  all  the  specific  and  group  agglutinins  acting  upon  its  own  type  and 
the  group  agglutinins  acting  upon  the  Coney  Island  and  normal  types,  leaving  the  specific  aggluti- 
nins for  types  Shiga  and  Mt.  Desert.  The  same  is  true  for  types  Shiga  and  Mt.  Desert  when  they  were 
used. 

Manila  paradysentery.  Mt.  De»ert  paradysentery. 

Japan  paradysentery.  —  and Atypical  paradysentery. 

The  absorption  method  simply  proves,  therefore,  that  when  one 
variety  of  bacteria  removes  all  agglutinins  for  a  second  the  agglutinins 
under  question  were  not  produced  by  that  second  variety. 

Loss  of  Capacity  in  Bacteria  to  be  Agglutinated  or  to  Absorb  Agglu- 
tinins Because  of  Growth  in  Immune  Sera. — The  loss  of  these  charac- 
teristics by  growth  in  sera  has  been  demonstrated  by  Marshall  and 
Knox.  The  experiments  of  Dr.  Collins  and  myself  are  recorded  be- 
cause they  were  undertaken  in  a  slightly  different  way  and  also  because 
a  certain  number  of  confirmatory  observations  are  of  value. 

The  maltose  fermenting  paradysentery  bacillus  of  Flexner  was 
grown  on  each  of  eleven  consecutive  days  in  fresh  bouillon  solutions 
of  the  serum  from  a  horse  immunized  through  oft-repeated  injections 
of  the  bacillus.  The  solutions  used  were  1.5,  4,  and  15  per  cent.  The 
serum  agglutinated  the  culture  before  its  treatment  in  dilutions  up  to 
1 : 800,  and  was  strongly  bactericidal  in  animals.  After  the  eleven 
transfers  the  culture  grown  in  the  15  per  cent,  solution  ceased  to  be 
agglutinated  by  the  serum  and  ceased  to  absorb  its  specific  agglutinins. 
The  cultures  grown  in  the  1.5  and  4  per  cent,  solutions  agglutinated 
well  in  dilutions  up  to  1 : 60  and  1 : 100  and  continued  to  absorb  agglu- 
tinins. The  recovery  of  the  capacity  to  be  agglutinated  was  very  slow 
when  the  culture  was  from  time  to  time  transplanted  on  nutrient  agar. 
After  growth  for  sixteen  weeks,  during  which  it  was  transplanted  forty- 
three  times,  it  agglutinated  in  dilutions  of  1 : 200.  The  culture  grown 
in  4  per  cent,  agglutinated  1:500,  and  the  one  in  1.5  per  cent.  1:800. 
This  diminution  and  final  cessation  of  development  of  agglutinable 
substance  in  bacteria  grown  in  a  serum  rich  in  agglutinin  and  immune 


184 


PRIXCIPLES  OF  BACTERIOLOGY 


bodies  is  interesting  both  as  showing  the  variation  of  the  bacteria  and 
as  one  means  of  adapting  themselves  to  resist  destruction,  since  the 
bacteria  which  ceased  to  produce  agglutinable  substance  probably  also 
produced  less  substance  with  affinity  for  other  antibodies.  This  inhi- 
bition of  the  production  of  agglutinable  substance  was  also  very 
noteworthy  in  the  case  of  pneumococci  grown  in  serum  media. 

Relation  between  Agglutinating  Bactericidal  Power.-  —  In  spite  of  proof 
to  the  contrary  many  good  observers  hold  to  the  belief  that  there  is 
some  relation  between  the  agglutinating  and  the  bactericidal  strength 
of  a  serum.  The  tests  we  carried  out  on  the  serum  of  a  number  of 
horses  showed  no  such  relation.  In  Fig.  73  are  recorded  a  num- 
ber of  comparative  tests  during  a  period  of  sixteen  months.  For  the 
tests  of  the  bactericidal  power  of  the  serum  we  are  indebted  to  Dr.  Mary 
E.  Goodwin.  She  also  showed  that  there  was  a  production  of  group. 
as  well  as  specific  immune  bodies  in  the  animals  receiving  prolonged 
injections.  The  results  of  her  experiments  will  be  published  later. 


1  and  2 


FiG.  73 
3  and  4    5  and  6    7  and  8    9  and  10    11  and  12    13  and  14    15  and  16 


1  1200 

Fatal 

|  1  100 

doses 

/'x 

\ 

1000 

protected 

/ 

\, 

N_ 

/\ 

900 

Iccserem 

/ 

A 

/  \ 

800 

4X) 

'   / 

\ 

700 

3.5 

/ 

\ 

1  .^ 

**** 

600 

3.0 

1 

\ 

,4 

^"~ 

500 

2.5 

j 

\~ 

400 

2.0 

/ 

^ 

\                  '• 

\ 

300 

1.5 

^*^- 

•^--—  ^ 

^ 

\           / 

\ 

200 

1.0 

^/ 

\      / 

\ 

^ 

100 

/ 

t 

00 

Relation  of  agglutinative  power  to  bactericidal.    Horse  injected  with  Manila  culture  over  a 
period  of  sixteen  months. 

—  Agglutination  index.  —  Bactericidal  index. 

•  Tests  dates. 

Variation  in  the  Agglutinating  Strength  of  a  Serum. — There  is  usually 
a  continued  increase  in  the  amount  of  agglutinin  in  the  blood  of  an 
infected  person  from  the  fourth  day  until  convalescence  and  then  a 
decrease.  At  times,  however,  there  is  a  marked  variation  from  day  to 
day,  so  that  it  may  be  abundantly  present  one  day  and  almost  absent 
the  next. 


PART  II. 

BACTERIA  PATHOGENIC  TO  MAN  INDIVIDUALLY 
CONSIDERED. 


CHAPTER  XVII. 

THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA. 

Historical  Notes. — The  specific  contagious  disease  which  we  now  call 
diphtheria  can  be  traced  back  under  various  names  to  almost  the  Homeric 
period  of  Grecian  history.  The  Greeks  believed  that  it  had  been  com- 
municated to  their  country  from  Egypt.  The  description  of  the  pharyn- 
geal  and  laryngeal  manifestations  of  this  disease  left  by  Aretseus  leaves 
no  doubt  that  it  was  of  diphtheria  that  he  wrote.  From  time  to  time 
during  the  following  centuries  we  hear  of  epidemics  both  in  Italy  and 
in  other  portions  of  the  civilized  world  which  indicate  that  the  disease 
never  absolutely  ceased.  The  disease  early  crossed  to  America,  and 
in  the  New  England  States  we  get  clear  accounts  of  its  ravages. 

In  1765  Home,  a  Scotchman,  tried  to  show  that  "croup"  and  pharyn- 
geal  diphtheria  were  different  diseases,  and  this  subject  remained 
under  controversy  until  it  was  settled,  through  bacteriological  examina- 
tions, that  while  most  cases  were  undoubtedly  diphtheria,  a  few  were 
not. 

In  1771  Bard,  an  American,  supported  the  opposite  theory  from  Home, 
considering  the  process  the  same  wherever  located.  His  observations 
.upon  diphtheria  were  very  important  and  accurate. 

In  1821  Bretonneau  published  his  first  essay  on  diphtheria  in  Paris 
and  gave  to  the  disease  its  present  name.  His  observations  were  so 
extensive  and  so  correct  that  little  advance  in  knowledge  took  place 
until  the  causal  relations  of  the  diphtheria  bacilli  and  their  associated 
micro-organisms  to  the  disease  began  to  be  recognized.  Since  then 
the  combined  clinical,  bacteriological,  and  pathological  studies  have 
sufficed  to  make  diphtheria  one  of  the  best  understood  of  diseases. 

The  Diphtheria!.  Bacillus. 

Discovery.— In  the  year  1883,  bacilli  which  were  very  peculiar  and 
striking  in  appearance  were  shown  by  Klebs  to  be  of  constant  occur- 
rence in  the  pseudomembranes  from  the  throats  of  those  dying  of  true 


BACTERIA  PATHOGENIC  TO  MAN 

epidemic  diphtheria.  One  year  later,  Loeffler  published  the  results  of 
a  very  thorough  and  extensive  series  of  investigations  on  this  subject. 
He  found  the  bacillus  described  by  Klebs  in  many  cases  of  throat  inflam- 
mations which  had  been  diagnosticated  as  diphtheria.  He  separated 
these  bacilli  from  the  other  bacteria  present  and  obtained  them  in  pure 
culture.  When  he  inoculated  the  bacilli  upon  the  abraded  mucous 
membrane  of  susceptible  animals  more  or  less  characteristic  pseudo- 
membranes  were  produced,  and  frequently  death  or  paralysis  followed 
with  characteristic  lesions. 

In  1887-88  further  studies  by  Loeffler,  Roux,  and  Yersin  added  to  the 
proof  of  the  dependence  of  diphtheria  on  this  bacillus.  It  was  found 
that,  while  no  other  forms  of  bacteria  were  constantly  met  with,  the 
diphtheria  bacilli  were  present  in  all  characteristic  cases  of  diphtheria, 
and  that  these  bacilli  possessed  the  morphological,  cultural,  and  patho- 
genic qualities  of  those  described  by  Klebs  and  Loeffler.  The  results 
of  these  investigations  have  since  been  confirmed  by  a  great  number 
of  combined  clinical  and  bacteriological  observations  both  in  animals 
and  human  beings. 

Human  Inoculation  Experiments. — A  very  instructive  accidental  ex- 
periment was  carried  out  under  our  observation  some  years  ago.  One 
of  the  investigators  unintentionally  drew  quite  a  quantity  of  a  bouillon 
culture  of  a  virulent  diphtheria  bacillus  into  the  throat,  and  two  days 
later  characteristic  diphtheria  of  a  serious  type  developed.  Similar 
accidents  have  happened  in  two  other  laboratories.  In  view  of  known 
facts,  we  are  now  justified  in  saying  that  the  name  diphtheria  should  be 
applied,  and  exclusively  applied,  to  that  acute  infectious  disease  usually 
associated  with  pseudomembranous  affection  of  the  mucous  membranes 
which  is  primarily  caused  by  the  bacillus  diphtheriae  of  Loeffler. 

Morphology. — When  cover-glass  preparations  made  from  the  cul- 
tures grown  on  blood  serum  are  examined,  the  diphtheria  bacilli  are 
found  to  possess  the  following  morphological  characteristics:  The 
diameter  of  the  bacilli  varies  from  0.3  to  0.8/*  and  the  length  from  1 
to  Q/Ji.  They  occur  singly  and  in  pairs  (see  Figs.  74  to  81)  and  very 
infrequently  in  chains  of  three  or  four.  The  rods  are  straight  or  slightly 
curved,  and  usually  are  not  uniformly  cylindrical  throughout  their 
entire  length,  but  are  swollen  at  the  end,  or  pointed  at  the  ends  and 
swollen  in  the  middle  portion.  The  average  length  of  the  bacilli  in 
pure  cultures  from  different  sources  frequently  varies  greatly,  arid  even 
from  the  same  culture  individual  bacilli  differ  much  in  their  size  and 
shape.  This  is  especially  true  when  the  bacilli  are  grown  in  association 
with  other  bacteria.  The  two  bacilli  of  a  pair  may  lie  with  their  long 
diameter  in  the  same  axis,  or  at  an  obtuse  or  an  acute  angle.  The 
bacilli  possess  no  spores,  but  have  in  them  highly  refractile  bodies, 
some  of  which  are  the  starting  point  for  new  bacilli. 

Staining.— The  Klebs-Loeffler  bacilli  stain  readily  with  ordinary 
aniline  dyes,  and  retain  fairly  well  their  color  after  staining  by  Gram's 
method.  With  Loeffler 's  alkaline  solution  of  methylene  blue,  and  to  a 
less  extent  with  other  weak  staining  solutions,  the  bacilli  from  blood- 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     187 

serum  cultures,  especially,  and  from  other  media  less  constantly,  stain 
in  an  irregular  and  extremely  characteristic  way.  (See  Fig.  74.)  The 
bacilli  do  not  stain  uniformly.  In  many  cultures  round  or  oval  bodies, 
situated  at  the  ends  or  in  the  central  portions,  stain  much  more  intensely 
than  the  rest  of  the  bacillus.  Sometimes  these  highly  stained  bodies 
are  thicker  than  the  rest  of  the  bacillus;  again,  they  are  thinner  and 
surrounded  by  a  more  slightly  stained  portion.  The  bacilli  stain  in 
this  peculiar  manner  at  a  certain  period  of  their  growth,  so  that  only 


FIG.  74 


FIG.  75 


FIG.  74.— One  of  very  characteristic  forms  of  diphtheria  bacilli  from  blood-serum  cultures,  showing 
clubbed  ends  and  irregular  stain,  x  1100  diameters.  Stain,  methylene  blue. 

FIG.  75.— Extremely  long  form  of  diphtheria  bacillus.  This  culture  has  grown  on  artificial  media 
for  four  years  and  produces  strong  toxin.  X  1100  diameters. 


FIG.  76 


FIG.  77 


FIG.  76.— Diphtheria  bacilli  characteristic  in  shapes,  but  showing  even  staining.  In  appearance 
similar  to  the  xerosis  bacillus.  X  1100  diameters.  Stain,  methylene  blue. 

FIG.  77.— Non- virulent  diphtheria  bacilli,  showing  stain  with  Neisser's  solutions,  supposed  to  be 
characteristic  of  virulent  bacilli.  Bodies  of  bacilli  in  smear,  faint  brown ;  points,  dark  blue. 

a  portion  of  the  organisms  taken  from  a  culture  at  any  one  time  will 
show  the  characteristic  staining.  In  old  cultures  it  is  often  difficult 
to  stain  the  bacilli,  and  the  staining,  when  it  does  occur,  is  frequently 
not  at  all  characteristic.  The  same  round  or  oval  bodies  which  take 
the  methylene  blue  more  intensely  than  the  remainder  of  the  bacillus 
are  brought  out  still  more  distinctly  by  the  Neisser  stain. 

The  Neisser  stain  is  carried  out  by  placing  the  cover-slip  smear  of 
diphtheria  or  other  bacilli  in  solution  No.  1  for  from  two  to  three  sec- 


BACTERIA  PATHOGENIC  TO  MAN 

onds,  and  then,  after  washing,  in  No.  2  for  from  three  to  five  seconds. 
The  bacilli  will  then  appear  either  entirely  brown  or  will  show  at  one 
or  both  ends  a  dark-blue,  round  body.  With  characteristic  diphtheria 
bacilli,  taken  from  a  twelve  to  eighteen  hours'  growth  on  serum,  nearly 
all  will  show  the  blue  bodies  (Fig.  76),  while  with  the  pseudotype  (Fig. 
83,  page  197),  to  be  described  hereafter,  few  will  be  seen. 
The  solutions  are  as  follows: 

No.   1. 

Alcohol  (96  per  cent. ) 20  parts. 

Met hylene  blue  (Griibler) 1  part. 

Distilled  water 950  parts. 

Acetic  acid  (glacial) 50     " 

No.  2. 

Bismarck  brown          ......         1  part. 

Boiling  distilled  water        .....     500  parts. 

The  Neisser  stain  has  been  advocated  in  order  to  separate  the  viru- 
lent from  the  non-virulent  bacilli,  without  the  delay  of  inoculating 
animals;  but  in  our  hands,  with  a  very  large  experience,  neither  the 
Neisser  stain  nor  other  stains,  such  as  the  modifications  of  the  Roux 
stain,  have  given  much  more  information  as  to  the  virulence  of  the 
bacilli  than  the  usual  methylene-blue  solution  of  Loeffler.  A  small 
percentage  of  virulent  bacilli  fail  to  take  the  Neisser  stain,  and  quite  a 
few  non-virulent  pseudodiphtheria  bacilli  show  the  dark  bodies.  In 
New  York  there  are  also  a  large  number  of  bacilli  which  seem  to  have 
all  the  staining  and  cultural  characteristics  of  the  virulent  bacilli,  and 
yet  are  non-virulent  in  the  sense  that  they  produce  no  specific  toxin. 
To  one  who  is  accustomed  to  the  Loeffler  stain  it  gives  as  much  informa- 
tion as  any  other,  as  to  the  specific  virulence  of  the  bacilli.  The  Neisser 
stain  as  well  as  some  others  will  undoubtedly  cause  the  examiner  to 
suspect  more  strongly  some  bacilli  of  being  virulent  than  the  Loeffler 
stain,  but  with  the  varieties  met  with  in  New  York  this  suspicion  is  as 
apt  to  be  wrong  as  right.  As  will  be  stated  more  fully  later,  nothing 
but  animal  inoculations  with  control  injections  of  antitoxin  will  separate 
bacilli  capable  of  producing  diphtheria  toxin  from  others. 

The  morphology  of  the  diphtheria  bacillus  varies  considerably  with 
different  culture  media  employed.  On  glycerin  agar  or  simple  nutrient 
agar  there  are  two  distinct  types.  One  grows  as  smaller  and,  as  a  rule, 
more  regular  forms  than  when  grown  on  serum  culture  media  (Fig.  78). 
The  other  type  shows  many  thick,  Indian-clubbed  forms  with  a  moderate 
number  of  segments.  Short,  spindle,  lancet,  or  club-shaped  forms, 
staining  uniformly,  are  all  observed.  The  bacilli  which  have  developed 
in  the  pseudomembranes  or  exudate  in  cases  of  diphtheria  resemble  in 
shape  young  bacilli  grown  on  blood  serum,  but  stain  more  evenly. 

Biology. — The  Klebs-Loeffler  bacillus  is  non-motile  and  non-lique- 
fying. It  is  aerobic.  It  grows  most  readily  in  the  presence  of  oxygen, 


THE  BACILLI'S  AM)  Till-:  HM'TI'.illOLOGY  OF  DIPHTHERIA     189 

but  also  without  it.  It  does  not  form  spores.  It  begins  to  develop,  but 
grows  slowly  at  a  temperature  of  20°  C.,  or  even  less.  It  attains 
its  maximum  development  at  37°  C.  In  old  cultures  in  fluid  media, 
Williams  has  observed  fusion  of  one  bacillus  with  another.  The  fused 
forms  live  the  longest. 

Ibxixtance  to  Heat,  Drying,  and  Chemicals. — Its  thermal  death  point 
with  ten  minutes'  exposure  is  about  60°  C.     Boiling  kills  in  one  minute. 


FIG.  78 


Fro.  79 


FIG.  78.— Diphtheria  bacilli  from  agar  culture.    X  1000  diameters. 

FIG.  79.— B.  diphtherias.  No.  31.  Forty-eight  hours'  agar  culture.    Thick,  medium-clubbed  rods  and 
moderate  number  of  segments.    One  year  on  artificial  culture  media.    X  1410  diameters. 


FIG.  <ij 


FIG.  81 


FIG.  80. — B.  diphtheria,  No.  57.  Forty-eight  hours'  agar  culture.  Many  segments;  long,  medium- 
clubbed  ends.  One  year  on  artificial  media.  X  1410  diameters. 

FIG.  81.— B.  diphtheria,  S.  Twenty-four  hours'  agar  culture.  Coccus  forms.  Segmented  granular 
forms  on  Loeffler's  serum.  Only  variety  found ;  cases  of  diphtheria  at  Children's  Home.  X  1410 
diameters. 

It  is  more  easily  destroyed  by  disinfectants  than  many  other  bacteria. 
In  the  dry  state  and  exposed  to  diffuse  light  diphtheria  bacilli 
usually  die  in  a  few  days  or  may  live  for  weeks  or  months;  when 
in  the  dark,  or  protected  by  a  film  of  mucus  or  albumin,  they  may 
live  for  even  longer  periods.  Thus  we  found  scrapings  from  a  dry 
bit  of  membrane  to  contain  vigorous  and  virulent  living  bacilli  for  a 
period  of  four  months  after  removal  from  the  throat,  and  if  the  mem- 
brane had  not  been  at  that  time  completely  used,  living  bacilli  could 


190 


BACTERIA  PATHOGENIC  TO  MAN 


FIG.  82 


probably  have  been  obtained  for  a  much  longer  period.  On  slate-  and 
lead-pencils,  as  well  as  on  paper  money,  they  may  live  for  several  weeks, 
while  on  coins  they  die  in  twelve  to  thirty-six  hours.  In  culture  media, 
when  kept  at  the  blood  heat,  they  usually  die  after  a  few  weeks;  but 
under  certain  conditions,  as  when  sealed  in  tubes  and  protected  from 
heat  and  light,  they  retain  their  virulence  for  years.  The  bacillus  is 
not  sensitive  to  cold,  for  we  found  it  to  retain  its  virulence  after  exposure 
for  two  hours  to  several  hundred  degrees  below  zero. 

Growth  on  Culture  Media. — BLOOD  SERUM,  especially  coagulated  in  the 
form  of  Loeffler's  mixture,  is  the  most  favorable  medium  for  the  growth 
of  the  diphtheria  bacillus,  and  is  used  particularly  for  diagnostic  pur- 
poses in  examining  cultures  from  the  throats  of  persons  suspected  of 
having  diphtheria.  For  its'  preparation,  see  p.  51.  If  we  examine 
the  growth  of  the  diphtheria  bacillus  in  pure  culture  on  blood  serum 
we  shall  find  at  the  end  of  from  eight  to  twelve  hours  small  colonies 

of  bacilli,  which  appear  as  pearl- 
gray,  whitish-gray,  or,  more  rarely, 
yellowish-gray,  slightly  raised  points. 
The  colonies  when  separated  from 
each  other  may  increase  in  forty- 
eight  hours  so  that  the  diameter 
may  be  one-eighth  of  an  inch.  The 
borders  are  usually  somewhat  un- 
even. The  colonies  lying  together 
become  confluent  and  fuse  into  one 
mass  when  the  serum  is  moist. 
During  the  first  twelve  hours  the 
colonies  of  the  diphtheria  bacilli 
are  about  equal  in  size  to  those  of 
the  other  pathogenic  bacteria  which 
colonies  of  diphtheria  bacim.  x  200  diameters.  are  often  present  in  the  throat;  but 

after  this  time  the  diphtheria  col- 
onies become  larger  than  those  of  the  streptococci  and  smaller  than 
those  of  the  staphylococci.  The  diphtheria  bacilli  in  their  growth  never 
liquefy  the  blood  serum. 

GROWTH  ON  AGAR. — On  1  per  cent,  slightly  alkaline,  plain  nutrient 
or  glycerin-agar  the  growth  of  the  diphtheria  bacillus  is  less  certain 
and  luxuriant  than  upon  blood  serum;  but  the  appearance  of  the  colo- 
nies when  examined  under  a  low-power  lens,  though  very  variable, 
is  often  far  more  characteristic.  (See  Fig.  82,  and  Fig.  44,  page  62.) 
For  this  reason  nutrient  agar  in  Petri  dishes  is  used  to  obtain  diph- 
theria bacilli  in  pure  culture.  The  diphtheria  bacillus  obtained  from 
cultures  which  have  developed  for  some  time  on  culture  media  grows 
well,  or  fairly  well,  on  suitable  nutrient  agar,  but  when  fresh  from 
pseudomembranes  one  prevalent  type  of  bacilli  grows  on  these  media 
with  great  difficulty,  and  the  colonies  develop  so  slowly  as  to  be 
frequently  covered  up  by  the  more  luxuriant  growth  of  other  bacteria 
when  present,  or  fail  to  develop  at  all. 


THE  BACILLUS  AXD  THE  BACTERIOLOGY  OF  DIPHTHERIA     191 

If  the  colonies  develop  deepen  the  substance  of  the  agar  they  are 
usually  round  or  oval,  and,  as  a  rule,  present  no  extensions;  but  if  near 
the  surface,  commonly  from  one,  but  sometimes  from  both  sides,  they 
spread  out  an  apron-like  extension,  which  exceeds  in  surface  area  the 
rest  of  the  colony.  When  the  colonies  develop  entirely  on  the  surface 
they  are  more  or  less  coarsely  granular,  and  usually  have  a  dark  centre 
and  vary  very  much  in  their  thickness.  The  colonies  from  some  are 
almost  translucent;  others  are  thick  and  almost  as  luxuriant  as  the 
staphylococcus.  The  edges  are  sometimes  jagged,  and  frequently  shade 
off  into  a  delicate  lace-like  fringe;  at  other  times  the  margins  are  more 
even  and  the  colonies  are  nearly  circular.  With  a  high-power  lens  the 
edges  show  sprouting  bacilli.  The  colonies  are  gray  or  grayish-white 
by  reflected  light,  and  pure  gray  with  an  olive  tint  by  transmitted  light. 

The  growth  of  the  diphtheria  bacillus  upon  agar  presents  certain 
peculiarities  which  are  of  practical  importance.  If  a  large  number  of 
the  bacilli  from  a  recent  culture  are  implanted  upon  a  properly  prepared 
agar  plate  a  certain  and  fairly  vigorous  growth  will  always  take  place. 
If,  however,  the  agar  is  inoculated  with  an  exudate  from  the  throat, 
which  contains  but  few  bacilli,  no  growth  whatever  may  occur,  while 
the  tubes  of  coagulated  blood  serum  inoculated  with  the  same  exudate 
contain  the  bacilli  abundantly.  Because  of  the  uncertainty,  therefore, 
of  obtaining  a  growth  by  the  inoculation  of  agar  with  bacilli  unaccus- 
tomed to  this  medium,  agar  is  not  a  reliable  medium  for  use  in  primary 
cultures  for  diagnostic  purposes.  A  mixture  composed  of  two  parts  of 
a  1.5  percent,  nutrient  agar  and  one  part  of  sterile  ascitic  fluid  makes 
a  medium  upon  which  the  bacillus  grows  much  more  luxuriantly,  but 
not  so  characteristically.  The  mixture  is  made  by  adding  ascitic  fluid , 
warmed  to  about  45°  to  50°  C.,  to  the  tubes  containing  the  melted  agar 
cooled  to  60°  C.  After  shaking,  the  Petri  plates  are  filled. 

Isolation  of  the  Diphtheria  Bacillus  from  Plate  Cultures. — Nutrient 
plain  or  glycerin-agar  is  the  medium  employed  to  get  by  plating  methods 
a  pure  culture  from  the  original  serum  tube.  The  agar  should  be  freshly 
melted  and  poured  in  the  Petri  dish  for  this  purpose.  After  it  has  hard- 
ened, the  layers  in  a  number  of  plates  are  streaked  across  with  bacteria 
from  colonies  on  the  serum  culture,  which  appear  in  size  and  color  like 
the  diphtheria  bacilli.  Other  plates  are  made  from  a  general  mixture 
of  all  the  bacteria,  selected,  as  a  rule,  from  the  drier  portion  of  the 
serum.  Other  plates  are  inoculated  from  the  pellicle  of  an  ascitic  broth 
culture.  The  plates  are  left  in 'the  incubator  for  about  sixteen  hours  at 
37°  C.  In  the  examination  of  the  plates  one  should  first  seek  for  typical 
colonies,  and  then  later  for  any  that  look  most  nearly  like  the  character- 
istic picture.  Diphtheria  colonies  are  very  apt  to  be  found  at  the  edges 
of  the  streaks  of  bacterial  growth. 

GROWTH  IN  BOUILLON. — The  diphtheria  bacilli  from  about  one- 
half  the  cultures  grow  readily  in  broth  slightly  alkaline  to  litmus;  the 
other  cultures  grow  very  feebly.  The  characteristic  growth  in  neutral 
bouillon  is  one  showing  fine  grains.  These  deposit  along  the  sides  and 
bottom  of  the  tube,  leaving  the  broth  nearly  clear.  A  few  cultures  in 


192  BACTERIA  PATHOGENIC  TO  MAN 

neutral  bouillon  and  many  in  alkaline  bouillon  produce  for  twenty- 
four  or  forty-eight  hours  a  more  or  less  diffuse  cloudiness,  and  fre- 
quently a  film  forms  over  the  surface  of  the  broth.  On  shaking  the 
tube  this  film  breaks  up  and  slowly  sinks  to  the  bottom.  This  film  is 
apt  to  develop  during  the  growth  of  cultures  which  have  long  been 
cultivated  in  bouillon,  and,  indeed,  after  a  time  the  entire  development 
may  appear  on  the  surface  in  the  form  of  a  friable  pellicle.  The 
diphtheria  bacillus  in  its  growth  causes  a  fermentation  of  meat-sugars 
and  glucose,  and  thus  if  these  are  present  changes  the  reaction  of  the 
bouillon,  rendering  it  distinctly  less  alkaline  within  forty-eight  hours,  and 
then,  after  a  variable  time,  when  all  the  fermentable  sugars  have  been 
decomposed,  more  alkaline  again  through  the  progressing  fermenta- 
tion of  other  substances.  Among  the  products  formed  by  its  growth 
is  the  diphtheria  toxin. 

GROWTH  IN  ASCITIC  OR  SERUM  BOUILLON. — Diphtheria  bacilli  grow 
well  in  this  medium,  even  when  first  removed  from  the  throat.  They 
almost  always  form  a  slight  pellicle  at  the  end  of  twenty-four  to  forty- 
eight  hours.  To  the  nutrient  bouillon  25  per  cent,  ascitic  fluid  or  blood 
serum  is  added.  This  culture  medium  is,  as  pointed  out  by  Williams, 
of  the  greatest  value  in  attempts  to  get  pure  cultures  of  the  diphtheria 
bacillus  from  solidified  serum  cultures  containing  few  bacilli. 

GROWTH  ON  GELATIN. — The  growth  on  this  medium  is  much  slower, 
more  scanty,  and  less  characteristic  than  that  on  the  other  media  men- 
tioned, on  account  of  the  lower  temperature  at  which  it  must  be  used. 

GROWTH  IN  MILK. — The  diphtheria  bacillus  grows  readily  in  milk, 
beginning  to  develop  at  a  comparatively  low  temperature  (20°  C.). 
Thus,  milk  having  become  inoculated  with  the  bacillus  from  some  cases 
of  diphtheria  may,  under  certain  conditions,  be  the  means  of  conveying 
infection  to  previously  healthy  persons.  The  milk  remains  unchanged 
in  appearance. 

Pathogenesis. — The  diphtheria  bacillus  is  pathogenic  for  guinea- 
pigs,  rabbits,  chickens,  pigeons,  small  birds,  and  cats;  also  in  a  lesser 
degree  for  dogs,  goats,  cattle,  and  horses,  but  hardly  at  all  for  rats  and 
mice.  In  spite  of  its  pathogenic  qualities  for  these  animals  true  diph- 
theria occurs  in  them  with  extreme  rarity.  As  a  rule,  supposed  diph- 
theritic inflammations  in  them  are  due  to  other  bacteria  which  cannot 
produce  the  disease  in  man. 

The  virulence  of  diphtheria  bacilli  from  different  sources,  as  meas- 
ured by  their  toxin  production  in  bouillon,  varies  enormously,  but  in 
ascitic  fluid  it  is  more  alike.  Thus  0.002  c.c.  of  a  forty-hour  bouillon 
culture  of  one  bacillus  will  kill  a  guinea-pig,  which  it  would  require  1  c.c. 
of  the  culture  of  another  bacillus  to  kill.  This  difference  frequently 
depends  on  the  unequal  growth  of  the  bacilli;  one  culture  having  fifty 
times  as  many  bacilli  as  the  other.  The  same  marked  variation  occurs 
in  the  amount  of  toxin  produced  by  different  bacilli  in  their  growth 
outside  of  the  body.  Moreover,  the  diphtheria  bacilli  differ  greatly 
in  the  tenacity  with  which  they  retain  their  virulence  when  grown 
outside  the  body.  The  bacillus  that  we  have  used  in  the  labora- 


'////.  HACILLUS  AXD  Till.   li.\ r TERIOLOGY  OF  DIPHTHERIA     193 

tory  of  the  Board  of  Health  lias  retained  its  virulence  almost  unaltered 
for  ten  years  in  bouillon  cultures.  Other  bacilli  have  lost  50  per  cent, 
of  their  virulence  after  being  kept  only  a  few  months.  The  passage  of 
diphtheria  bacilli  through  the  bodies  of  susceptible  animals  does  not 
increase  their  toxin  production  to  any  considerable  extent. 

At  the  autopsy  of  animals  dying  from  the  poisons  produced  by  the 
bacilli,  the  characteristic  lesions  described  by  Loeffler  are  found.  At 
the  seat  of  inoculation  there  is  a  grayish  focus  surrounded  by  an  area 
of  congestion;  the  subcutaneous  tissues  for  some  distance  around  are 
oedematous;  the  adjacent  lymph  nodes  are  swollen;  and  .the  serous 
cavities,  especially  the  pleura  and  the  pericardium,  frequently  contain 
an  excess  of  fluid,  usually  clear,  but  at  times  turbid;  the  lungs  are  gen- 
erally congested.  In  the  organs  are  found  numerous  smaller  and  larger 
masses  of  necrotic  cells,  which  are  permeated  by  leukocytes.  The 
heart  and  certain  voluntary  muscular  fibres  and  tissues  of  nerves  usually 
show  degenerative  changes.  Occasionally  there  is  fatty  degeneration 
of  the  liver  and  kidneys.  The  number  of  leukocytes  in  the  blood  is 
increased.  From  the  area  surrounding  the  point  of  inoculation  viru- 
lent bacilli  may  be  obtained,  but  in  the  internal  organs  they  are  only 
occasionally  found,  unless  an  enormous  number  of  bacilli  have  been 
injected.  Paralysis,  commencing  usually  in  the  posterior  extremities 
and  then  gradually  extending 'to  the  whole  body  and  causing  death  by 
paralysis  of  the  heart  or  respiration,  is  also  produced  in  many  cases  in 
which  the  inoculated  animals  do  not  succumb  to  a  too  rapid  intoxication. 
In  a  number  of  animals  we  have  seen  recovery  take  place  three  to  six 
weeks  after  the  onset  of  the  paralysis.  The  occurrence  of  these  par- 
alyses, following  the  introduction  of  the  diphtheria  bacilli,  completes 
the  resemblance  of  the  experimental  disease  to  the  natural  malady  in 
man. 

Diphtheria  Toxin. — It  is  evident  that  a  micro-organism  which,  when 
injected  subcutaneously,  destroys  the  life  of  susceptible  animals  and 
produces  such  marked  anatomical  changes  in  the  internal  organs,  while 
it  is  found  only  at  or  near  the  point  of  inoculation,  must  owe  its  patho- 
genic power  to  the  formation  of  a  poison  which,  being  absorbed,  gives 
rise  to  toxaemia  and  death.  This  poison  or  toxin  has  been  partially 
isolated  by  Roux  and  Yersin,  and  others,  by  filtration  through  porous 
porcelain  from  cultures  of  the  living  bacilli.  It  has  not  yet  been  suc- 
cessfully analyzed,  so  that  its  chemical  composition  is  unknown,  but 
it  has  many  of  the  properties  of  proteid  substances,  and  can  well  be 
designated  by  the  term  active  proteid.  The  poison  produced  is 
probably  composed  of  a  mixture  of  several  nearly  related  toxins. 
Diphtheria  toxin  is  totally  destroyed  by  boiling  for  five  minutes,  and 
loses  some  95  per  cent,  of  its  strength  when  exposed  to  75°  C.  for  the 
same  time;  73°  C.  destroys  only  about  85  per  cent,  and  60°  very  little. 
Lower  temperatures  only  alter  it  very  gradually.  Kept  from  light  and 
air  and  in  cold  storage  it  deteriorates  very  slowly.  The  views  of 
Khrlich  and  Madsen  as  to  the  nature  of  toxins  will  be  considered 
in  the  chapter  under  its  relations  to  antitoxin. 

13 


194  BACTERIA  PATHOGENIC  TO  MAN 

The  Production  of  Toxin  in  Culture  Media. — The  artificial  production 
of  toxin  in  cultures  of  the  diphtheria  bacillus  has  been  found  to  depend 
upon  definite  conditions,  which  are  of  practical  importance  in  obtaining 
toxin  for  the  inoculation  of  horses,  and  also  of  theoretical  interest  in 
explaining  why  cases  of  apparently  equal  local  severity  have  such 
different  degrees  of  toxic  absorption.  The  researches  of  Roux  and 
Yersin  laid  the  foundation  of  our  knowledge.  Their  investigations 
have  been  continued  by  Theobald  Smith,  Spronck,  ourselves,  and 
others.  After  an  extensive  series  of  investigations  we  (Park  and  Wil- 
liams) came  to  the  following  conclusions:  Toxin  is  produced  by  fully 
virulent  diphtheria  bacilli  at  all  times  during  their  life  when  the  condi- 
tions are  favorable.  Under  less  favorable  conditions  some  bacilli  are 
able  to  produce  toxin  while  others  are  not;  or  it  may  be  that  some 
conditions  favor  some  bacilli  while  they  are  deleterious  to  others.  Diph- 
theria bacilli  may  find  conditions  suitable  for  luxuriant  growth,  but 
unsuitable  for  the  production  of  toxin.  The  requisite  conditions  for 
good  development  of  toxin,  as  judged  by  the  behavior  of  a  number  of 
cultures,  are  a  temperature  from  about  35°  to  36°  C.,  a  suitable  culture 
medium,  such  as  a  2  per  cent,  peptone  nutrient  bouillon  of  an  alkalinity 
which  should  be  about  8  c.c.  of  normal  soda  solution  per  litre  above 
the  neutral  point  to  litmus,  and  prepared  from  a  suitable  peptone  (Witte) 
and  meat.  The  culture  fluid  should  be*  in  comparatively  thin  layers 
and  in  large-necked  Erlenmeyer  flasks,  so  as  to  allow  of  a  free  access 
of  air.  The  greatest  accumulation  of  toxin  in  bouillon  is  after  a  dura- 
tion of  growth  of  the  culture  of  from  five  to  ten  days,  according  to  the 
peculiarities  of  the  culture  employed.  At  a  too  early  period  toxin  has 
not  sufficiently  accumulated;  at  a  too  late  period  it  has  begun  to  degen- 
erate. In  our  experience  the  amount  of  muscle-sugar  present  in  the 
meat  makes  no  appreciable  difference  in  the  toxin  produced  when  a 
vigorously  growing  bacillus  is  used,  so  long  as  the  bouillon  has  been 
made  sufficiently  alkaline  to  prevent  the  acid  produced  by  the  fer- 
mentation of  the  sugar  from  producing  in  the  bouillon  an  acidity  suffi- 
cient to  inhibit  the  growth  of  the  bacilli.  If  the  sugar  does  interfere 
this  can  be  prevented  by  its  previous  destruction  through  the  fermenta- 
tion caused  by  the  growth  of  the  colon  bacilli.  After  the  fermentation 
0.1  per  cent,  of  glucose  should  be  added.  Besides  the  sugar  and  allied 
bodies  in  the  meat  there  are  other  substances  whose  nature  is  unknown, 
which  hinder  or  aid  a  full  growth  of  the  bacilli  or  production  of  toxin. 
This  is  true  of  bouillon  made  directly  from  fresh  meat,  fermented  meat, 
or  meat  extracts.  With  the  meat  as  we  obtain  it  in  New  York,  we  get 
better  results  with  unfermented  meat  than  with  fermented.  In  Boston, 
with  the  same  bacillus,  Smith  gets  more  toxin  from  the  fermented  bouil- 
lon. Instead  of  colon  bacilli,  yeast  may  be  added  to  the  soaking  meat, 
which  is  allowed  to  stand  at  about  25°  C. 

Under  the  best  conditions  we  can  devise,  toxin  begins  to  be  produced 
by  bacilli  from  some  cultures  when  freshly  sown  in  bouillon  some  time 
during  the  first  twenty-four  hours;  from  other  cultures,  for  reasons  not 
well  understood,  not  for  from  two  to  four  days.  In  neutral  bouillon 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     195 

the  culture  fluid  frequently  becomes  slightly  acid  and  toxin  production 
may  be  delayed  for  from  one  to  three  weeks.  The  greatest  accumula- 
tion of  toxin  is  on  the  fourth  day,  on  the  average,  after  the  rapid  pro- 
duction of  toxin  has  commenced.  After  that  time  the  number  of  living 
bacilli  rapidly  diminishes  in  the  culture,  and  the  conditions  for  those 
remaining  alive  are  not  suitable  for  the  rapid  production  of  toxin.  As 
the  toxin  is  not  stable,  the  deterioration  taking  place  in  the  toxin  already 
produced  is  greater  than  the  amount  of  new  toxin  still  forming. 

Bacilli,  when  repeatedly  transplanted  from  bouillon  to  bouillon, 
gradually  come  to  grow  on  the  surface  only.  This  characteristic  seems 
to  aid  in  the  development  of  toxin. 

The  relations  of  toxin  to  antitoxin  will  be  considered  later  in  this 
chapter. 

Diphtheria-like  Bacilli  Not  Producing  Diphtheria   Toxin. — In  the  tests 
of  the  bacilli  obtained  from  hundreds  of  cases  of  suspected  diphtheria 
which  have  been  carried  out  during  the  past  ten  years  in  the  labora- 
tories of  the  Health  Department  of  New  York  City,  in  over  95  per 
cent,  of  cases  the  bacilli  derived  from  exudates  or  pseudomembranes 
and   possessing   the  characteristics  of  the  Loeffler  bacilli  have  been 
found  to  be  virulent,  that  is,  producers  of  diphtheria  toxin.    But  there 
are,  however,  in  inflamed  throats  as  well  as  in  healthy  throats,  either 
alone    or    associated    with    the    virulent    bacilli,    occasionally    bacilli 
which,    though    morphologically    and    in    their    behavior    on   culture 
media  identical  with  the  Klebs-Loeffler   bacillus,   are  yet  producers, 
at  least  in  artificial  culture  media  and  the  usual  test  animals,  of  no  diph- 
theria toxin.    Between  bacilli  which  produce  a  great  deal  of  toxin  and 
those  which  produce  none  we  find  a  few  of  minor  grades  of  virulence. 
We  believe,  therefore,  in  accordance  with  Roux  and  Yersin  these  non- 
virulent  bacilli  should  be  considered  as  possibly  attenuated  varieties 
of  the  diphtheria  bacillus  which  have  lost  their  power  to  produce  diph- 
theria toxin.    These  observers,  and  others  following  them,  have  shown 
that  the  virulent  bacilli  can  be  artificially  attenuated;  but  the  reverse  has 
not  been  proven  that  bacilli  which  produce  no  specific  toxin  have  later 
been  found  to  develop  it.    In  our  experience  some  cultures  hold  their  viru- 
lence even  when  grown  at  41°  C.  for  a  number  of  months,  while  others 
lose  it  more  quickly.     Diphtheria-like  bacilli  are  also  found  which 
resemble   diphtheria  bacilli  very  closely  except  in  toxin  production, 
but  differ  in  one  or  more  particulars.     Both  these  and  the  character- 
istic non-virulent  bacilli  are  found  occasionally  upon  all  the  mucous 
membranes!  both  when  inflamed  and  when  apparently  normal.    From 
varieties  of  this  sort  having  been  found  in  a  number  of  cases  of  the 
condition  known  as  xerosis  conjunctive,  these  bacilli  are  often  called 
xerosis  bacilli.    Under  this  name  different  observers  have  placed  bacilli 
identical  with  the  diphtheria  bacilli  and  others  differing  quite  mark- 
edly from  them. 

Bacilli  Virulent  to  Guinea-pigs  in  Spite  of  Producing  no  Diphtheria 
Toxin. — These  bacilli  are  obtained  fairly  frequently  from  normal  or 
slightly  inflamed  throats  and  may  be  only  slightly  pathogenic  in 


196  BACTERIA  PATHOGENIC  TO  MAN 

guinea-pigs,  or  they  may  kill,  as  we  have  found  in  a  number  of 
instances,  in  doses  of  2  to  5  c.c.  subcutaneously  or  intraperitoneally 
injected.  Animals  are  not  protected  by  diphtheria  antitoxin  from  the 
action  of  these  bacilli.  At  autopsy  the  bacilli  are  usually  found  more 
or  less  abundantly  in  the  blood  and  internal  organs.  The  fact  that 
large  injections  of  antitoxic  serum  hastens  the  death  of  guinea-pigs 
injected  with  these  bacilli,  has  given  rise  to  the  notion  that  injections 
of  antitoxin  might  be  dangerous  in  persons  in  whose  throats  these 
bacilli  were  present,  either  as  saprophytes  or,  possibly,  as  inciters  of 
slight  disease.  It  is  not  the  antitoxin,  but  the  serum,  which  in  large 
doses  injures  the  vitality  of  the  guinea-pigs  and  so  slightly  hastens 
death.  Any  serum  has  the  effect.  These  bacilli  were  first  described  by 
Miss  Davis1  from  my  laboratory  and  later  by  Dr.  Alice  Hamilton  in 
1904.  In  my  judgment  they  present  no  more  reason  to  avoid  giving 
antitoxin  than  do  the  streptococci  and  influenza  bacilli.  When  patho- 
genic in  man  they  are  usually  only  feebly  so. 

Location  of  Diphtheritic  Inflammations  and  Virulence  of  Bacilli. — Viru- 
lent bacilli  produce  and  are  found  not  only  in  pseudomembranous 
inflammations  of  the  fauces,  larynx,  and  nasal  cavities,  but  also  occa- 
sionally in  membranous  affections  of  the  skin,  vagina,  rectum,  con- 
junctiva, nose,  and  ear  (simple  membranous  rhinitis  and  otitis  media). 
From  the  severity  of  an  isolated  case  the  virulence  of  the  bacilli  cannot 
be  accurately  determined.  The  most  virulent  bacillus  we  have  ever 
found  was  obtained  from  a  mild  case  of  diphtheria  simulating  tonsillitis. 
Another  case,  however,  infected  by  the  bacillus  proved  to  be  very  severe. 
In  localized  epidemics  the  average  severity  of  the  cases  probably  indi- 
cates roughly  the  virulence  of  the  bacillus  causing  the  infection,  as  here 
the  individual  susceptibility  of  the  different  persons  infected  would, 
in  all  likelihood,  when  taken  together,  be  similar  to  that  of  other  groups; 
but  even  in  this  instance  special  conditions  of  climate,  food,  or  race 
may  influence  certain  localities.  Moreover,  the  bacteria  associated 
with  the  diphtheria  bacilli,  and  which  are  liable  to  be  transmitted 
with  them,  may  influence  the  severity  of  and  the  complications  arising 
in  the  cases.  It  must  be  remembered  that  bacilli  of  like  toxic  power 
may  differ  in  their  liability  to  infect.  Virulence  has  thus  two  distinct 
meanings  when  used  to  describe  diphtheria  bacilli. 

Virulent  Bacilli  in  Healthy  Throats. — Fully  virulent  bacilli  have  fre- 
quently been  found  in  healthy  throats  of  persons  who  have  been  brought 
in  direct  contact  with  diphtheria  patients  or  infected  clothing  without 
contracting  the  disease.  It  is,  therefore,  apparent  that  infection  in 
diphtheria,  as  in  other  infectious  diseases,  requires  not  only  the  pres- 
ence of  virulent  bacilli,  but  also  a  susceptibility  to  the  disease,  which 
may  be  inherited  or  acquired.  Among  the  predisposing  influences 
which  contribute  to  the  production  of  diphtheritic  infection  may  be 
mentioned  the  breathing  of  foul  air  and  living  in  overcrowded  and 
ill-ventilated  rooms,  poor  food,  certain  diseases,  more  particularly 

1  Medical  News,  April  29,  1899. 


Till-    HACILLUS  AXD  THE  BACTERIOLOGY  OF  DIPHTHERIA     197 

catarrhal  inflammations  of  the  mucous  membranes,  and  depressing 
conditions  generally.  Under  these  conditions  an  infected  mucous  mem- 
brane may  become  susceptible  to  disease.  In  connection  with  Beebe 
(1894)  I  made  an  examination  of  the  throats  of  330  healthy  persons 
who  had  not  come  in  contact,  so  far  as  known,  with  diphtheria,  and 
we  found  virulent  bacilli  in  8  only,  2  of  whom  later  developed  the  dis- 
ease. In  24  of  the  330  healthy  throats  non-virulent  bacilli  or  attenuated 
forms  of  the  diphtheria  bacillus  were  found.  Very  similar  observations 
have  been  made  by  others  in  many  widely  separated  countries. 

Persistence  of  Diphtheria  Bacilli  in  the  Throat. — The  continued  pres- 
ence of  virulent  diphtheria  bacilli  in  the  throats  of  patients  who 
have  recovered  from  the  disease,  and  after  the  disappearance  of  the 
exudate,  has  been  repeatedly  demonstrated.  Beebe  and  I  found  that 
in  304  of  605  consecutive  cases  the  bacilli  disappeared  within  three 
days  after  the  disappearance  of  the  pseudomembrane ;  in  176  cases 
they  persisted  for  seven  days,  in  64  cases  for  twelve  days,  in  36  cases 
for  fifteen  days,  in  12  cases  for  three  weeks,  in  4  cases  for  four  weeks, 
and  in  2  cases  for  nine  weeks.  Since  then  I  have  met  with  a  case  in 
which  they  persisted  with  full  virulence  for  eight  months.  Later  figures 
agree  substantially  with  these. 

Pseudodiphtheria  Bacilli.— Besides  the  typical  bacilli  which  produce 
diphtheria  toxin  and  those  which  do  not,  but  which,  so  far  as  we  can 
determine,  are  otherwise  identical  Avith  the  Loeffler  bacillus,  there  are 
other  bacilli  found  in  positions  similar  to 
those  in  which  diphtheria  bacilli  abound, 
which,  though  resembling  these  organisms 
in  many  particulars,  yet  differ  from  them  as 
a  class  in  others  equally  important.  The 
variety  most  prevalent  is  rather  short,  plump, 
and  more  uniform  in  size  and  shape  than 
the  true  Loeffler  bacillus  (Fig.  83).  On 
blood-serum  their  colony  growth  is  very 
similar  to  that  of  the  diphtheria  bacilli. 
The  great  majority  of  them  in  any  culture 
show  no  polar  granules  when  stained  by  the 

x^   .  .  J  Pseudodiphtheria  bacilli. 

Neisser  method,  and  stain  evenly  through- 
out with  the  alkaline  methylene-blue  solution.  They  do  not  produce 
acid  by  the  fermentation  of  glucose,  as  do  all  known  virulent  and  many 
non-virulent  diphtheria  bacilli;  therefore,  there  is  no  increase  in  acidity 
in  the  bouillon  in  which  they  are  grown  during  the  first  twenty-four 
hours  from  the  fermentation  of  the  meat-sugar  regularly  present.  They 
are  found  in  varying  abundance  in  different  localities  in  New  York 
City,  in  about  1  per  cent,  of  the  normal  throat  and  nasal  secretions, 
and  seem  to  have  now  at  least  no  connection  with  diphtheria;  whether 
they  were  originally  derived  from  diphtheria  bacillus  is  doubtful;  they 
certainly  seem  to  have  no  connection  with  it  now.  They  never  pro- 
duce diphtheria  toxin,  and  to  them  properly  has  been  applied  the  name 
psendodiphtheria  bacilli.  In  bouillon  they  grow,  as  a  rule,  less  luxuri- 


198  BACTERIA  PATHOGENIC  TO  MAN 

antly  than  the  diphtheria  bacilli.  Some  of  the  varieties  of  the  pseudo- 
diphtheria  bacilli  are  as  long  as  the  shorter  forms  of  the  virulent  bacilli. 
When  these  are  found  in  cultures  from  cases  of  suspected  diphtheria 
they  may  lead  to  an  incorrect  diagnosis.  The  Neisser  staining  method 
is  of  value  here,  but,  unfortunately,  the  absence  of  the  stained  bodies 
in  smears  of  mixed  cultures  is  not  a  sufficient  ground  to  exclude  the 
possibility  of  their  being  true  diphtheria  bacilli.  There  are  also  some 
varieties  which  resemble  the  short  pseudobacilli  in  form  and  staining, 
but  which  produce  acid  in  glucose  bouillon.  These  bacilli  are  found 
occasionally  in  all  countries  where  search  has  been  made  for  them. 
It  may  be  added  here  that  no  facts  have  come  to  light  which  indicate 
that  bacilli  which  do  not  produce  diphtheria  toxin  in  animals  ever  pro- 
duce it  in  man.  It  must  also  be  borne  in  mind,  however,  that  such 
proof  is  necessarily  very  difficult  to  obtain. 

Mixed  Infection  in  Diphtheria. — Virulent  diphtheria  bacilli,  however, 
are  not  the  only  bacteria  present  in  human  diphtheria.  Various  cocci, 
more  particularly  streptococci,  staphylococci,  and  pneumococci,  are 
also  found  associated  with  Loeffler's  bacilli  in  diphtheria,  playing  an 
important  part  in  the  disease  and  leading  often  to  serious  complications 
(sepsis  and  bronchopneumonia).  Indeed,  the  prognosis  in  a  case  of 
diphtheria  is  now  judged  to  be  graver,  other  things  being  equal,  accord- 
ing to  the  degree  to  which  other  pathogenic  bacteria  influence  the 
course  of  the  disease.  These  cases  of  so-called  mixed  infection  in  diph- 
theria have  within  recent  years  attracted  considerable  attention,  and 
have  been  the  subject  of  a  number  of  animal  experiments.  Though 
the  results  of  these  investigations  so  far  have  been  somewhat  indefinite, 
they  would  seem  to  indicate  that  when  other  bacteria  are  associated 
with  the  diphtheria  bacilli  they  mutually  assist  one  another  in  their 
attacks  upon  the  mucous  membrane,  the  streptococcus  being  particu- 
larly active  in  this  respect,  often  opening  the  way  for  the  invasion  of 
the  Loeffler  bacillus  into  the  deeper  tissues  or  supplying  needed  condi- 
tions for  the  development  of  its  toxin.  Thus  diphtheria  is  not  always 
a  primary,  but  often  a  secondary  disease,  following  some  other  infec- 
tion, as  measles  or  scarlet  fever.  In  most  fatal  cases  of  bronchopneu- 
monia following  laryngeal  diphtheria  we  find  not  only  abundant  pneu- 
mococci or  streptococci  in  the  inflamed  lung  areas,  but  also  in  the  blood 
and  tissues  of  the  organs.  As  these  septic  infections  due  to  the  pyo- 
genic  cocci  are  in  no  way  influenced  by  the  diphtheria  antitoxin,  they 
frequently  are  the  cause  of  the  fatal  termination.  Other  bacteria  cause 
putrefactive  changes  in  the  exudate,  producing  alterations  in  color 
and  offensive  odors. 

Pseudomembranous  Exudative  Inflammations  Due  to  Bacteria  other 
than  the  Diphtheria  Bacilli.— The  diphtheria  bacillus,  though  the  most 
usual,  is  not  the  only  micro-organism  that  is  capable  of  producing 
pseudomembranous  inflammations.  There  are  numerous  bacteria 
present  almost  constantly  in  the  throat  secretions,  which,  under  cer- 
tain conditions,  can  cause  local  lesions  very  similar  to  those  in  the  less- 
marked  cases  of  true  diphtheria.  The  streptococcus  and  pneumo- 


Till-:  HACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     199 

coccus  are  the  two  forms  most  frequently  found  in  these  cases,  but 
there  are  also  others  which,  under  suitable  conditions,  take  an  active 
part  in  producing  this  form  of  inflammation.  Among  these  is  a  long, 
slender  bacillus  which  is  occasionally  found  in  great  abundance  in  the 
middle  layers  of  pseudomembranes  when  the  diphtheria  bacillus  is 
absent.  This  bacillus  was  first  described  by  Vincent.1  It  does  not 
grow  readily  on  artificial  media  and  is  not  pathogenic  in  animals. 
From  its  presence  in  the  ulcerated  processes  and  false  membrane  of  a 
number  of  cases,  it  is  believed  to  have  some  causal  relation  to  them. 
This  bacillus  does  not  grow  on  the  serum  media,  so  that  the  diagnosis 
must  be  made  from  smears. 

These  cases  show  many  of  the  local  appearances  of  true  diphtheria, 
the  superficial  necrosis  of  the  epithelium,  the  membrane  of  the  glandu-? 
lar  swellings.  The  pseudomembranes  may  persist  for  from  one  to 
two  weeks,  or  even,  in  exceptional  cases,  longer.  This  bacillus  is  appar- 
ently frequently  present  in  the  normal  throat,  and  is  probably  only 
able  under  certain  favorable  conditions,  such  as  the  influence  of  syphilis, 
to  produce  lesions.  Nerve  degeneration  does  not  follow  an  attack. 

The  pseudomembranous  angina  accompanying  scarlet  fever,  and 
to  a  less  extent  other  diseases,  may  not  show  the  presence  of  diphtheria 
bacilli,  but  only  the  pyogenic  cocci,  especially  streptococci,  or,  more 
rarely,  some  varieties  of  little-known  bacilli.  The  deposit  covering  the 
inflamed  tissues  in  these  non-specific  cases  is,  it  is  true,  usually  but  not 
always,  rather  an  exudate  than  a  true  pseudomembrane.  The  majority 
of  these  cases,  however,  are  mild  affections,  being  only  of  importance 
in  adding  to  the  severity  of  the  disease  which  they  complicate.  An 
exception  should  be  made  when  the  larynx  is  affected,  as  here  the  lungs 
are  often  secondarily  involved.  The  bacteria  which  occur  in  false 
diphtheria  are  streptococci,  staphylococci,  diplococci,  and  sometimes 
pseud odiphtheria  bacilli  or  bacilli  which  are  morphologically  and  cul- 
turally distinct  from  the  diphtheria  bacilli. 

Persistence  of  Varieties  of  the  Bacillus  Diphtherias  and  of  Diphtheria- 
like  Bacilli. — The  fact  that  there  are  many  varieties  of  the  diphtheria 
and  diphtheria-like  bacillus  has,  we  think,  been  fully  established. 

But  that  such  varieties  are  true  sub-species  with  constant  charac- 
teristics, one  variety  not  changing  into  another  of  the  established  forms, 
has  not  been  generally  accepted.  On  the  contrary,  of  late  the  idea 
seems  to  be  gaining  ground  among  some  investigators  that  all  of  the 
various  forms  of  diphtheria-like  bacilli  are  the  result  of  more  or  less 
transitory  variations  of  the  same  species,  and  hence  that  the  virulent 
forms  are  the  result  of  a  rapid  adaptation  to  environment  and  conse- 
quent pathogenesis  of  the  non-virulent  forms,  both  typical  and  atypical. 

This  question  of  the  relationship  of  the  specifically  virulent  diph- 
theria bacillus  to  non-virulent,  diphtheria-like  bacilli  has  been  dis- 
cussed since  1887.  It  is  certainly  theoretically  possible  that  the  non- 
virulent  forms  have  been  derived  from  virulent  forms.  Whether  or 

'  Annales  de  1'Institut  Pasteur,  August,  1899. 


200  BACTERIA  PATHOGENIC  TO  MAN 

not  this  is  true  is  an  interesting  problem  for  discussion,  but  has  little 
practical  importance.  The  possibility  of  the  non-toxin  producing 
forms  readily  assuming  their  power  to  produce  toxin  is  of  the  greatest 
importance,  and  if  true  would  cause  us  to  change  our  present  methods 
of  trying  to  prevent  the  spread  of  diphtheria. 

Until  1896  no  one  had  brought  forward  evidence  to  show  that  fully 
non-virulent  forms  could  be  made  virulent.  In  this  year  Trump1  states 
that  he  converted  a  non-virulent  acid,  producing  bacillus  into  one 
capable  of  killing  guinea-pigs  with  all  the  symptoms  of  true  diphtheria, 
by  successive  passages  through  guinea-pigs  plus  a  non-fatal  dose  of 
diphtheria  toxin.  Hewlett  and  Knight2  state  (1897)  that  they  changed 
a  typical  virulent  diphtheria  bacillus  into  a  non-virulent  bacillus  of 
the  pseudo  type  by  heating  for  seventeen  hours  at  45°  C.  They  only 
succeeded  with  one  culture,  though  they  tried  others.  They  say  also 
that  they  changed  a  non-acid  pseudodiphtheria  bacillus  into  a  typical 
virulent  diphtheria  bacillus  by  culture  and  passage  through  guinea- 
pigs.  They  obtained  similar  but  not  such  marked  results  with  other 
cultures. 

Richmond  and  Salter3  (1898)  and  Salter4  (1899)  state  that  they  have 
changed  five  pseudodiphtheria  bacilli  into  typical  diphtheria  bacilli 
specifically  virulent  for  guinea-pigs  by  passage  through  a  number  of 
goldfinches. 

Bergey5  was  not  able  to  give  virulence  to  non-virulent  forms,  neither 
did  he  find  that  these  latter  gave  immunity  against  the  former;  for 
these  reasons  he  considers  them  distinct  members  of  a  large  group  of 
bacilli  at  the  head  of  which  stands  the  diphtheria  bacillus. 

In  the  work  of  Wesbrook,  Wilson,  and  McDaniel,6  on  Varieties  of 
Bacillus  Diphtheria,  the  study  is  based  upon  the  morphology  of  the 
individual  bacillus  found  in  smears  of  throat  cultures  and  pure  cultures. 
They  give  as  a  reason  for  the  study  of  the  individual  bacillus  that  in 
"pure  cultures  in  most  instances,  especially  where  they  have  been 
derived  from  typical  clinical  cases  of  diphtheria,  it  is  the  exception 
to  get  even  a  moderate  degree  of  uniformity  in  the  general  shape,  size, 
staining  reactions,  etc.,  of  the  individual  bacilli;  whilst  to  get  com- 
plete uniformity  is  not  to  be  hoped  for,"  and  therefore  each  culture  is 
probably  a  mixture  of  several  varieties  having  been  derived  from  several 
parents.  This  seems  to  us  to  be  probably  an  erroneous  conclusion. 
They  make  a  provisional  classification  based  upon  the  morphology 
of  the  individual  bacilli,  into  three  groups,  called  granular,  barred, 
and  solid,  two  of  the  groups  into  seven  types  and  the  other  into  five, 
two  of  the  types  corresponding  with  those  in  the  other  groups  not  hav- 
ing been  seen.  In  a  study  of  the  types  found  in  the  smears  from  a 
series  of  direct  cultures  derived  from  clinical  cases  of  diphtheria  the 

Centralblatt  fiir  Bakt.,  etc.,  1896,  Band  xx.  p.  721. 

Trans,  of  the  Brit.  Jnst.  of  Prev.  Med.,  1897, 1st  series. 

Guy's  Hospital  Reports,  1898. 

Trans,  of  the  Jenner  lust,  of  Prev.  Med.,  1899. 

Pub.  of  the  Univ.  of  Penn.,  1898,  new  series,  No.  4  (other  references). 

Transactions  of  the  Association  of  American  Physicians,  1900. 


Til !'-   HACILLl'S  A \ D  THE  BA C TERIOLOG Y  OF  DIPH THERIA     20 1 

authors  state  that  there  is  generally  a  sequence  of  types  in  the  varia- 
tions which  appear  throughout  the  course  of  the  disease,  the  granular 
types  being  the  most  predominating  at  the  outset  of  the  disease,  and 
these  giving  place  wholly  or  in  part  to  the  barred  and  solid  types  shortly 
before  the  disappearance  of  diphtheria-like  organisms. 

The  inference  drawn  from  this  work  is  that  the  diphtheria  bacillus 
may  be  rather  easily,  especially  in  the  throat,  converted  into  non- 
granular,  solidly  staining  forms  of  the  "  pseudodiphtheria"  type,  and 
that  the  converse  may  occur,  and  that  therefore  all  diphtheria-like 
bacilli  must  be  considered  a  possible  source  of  danger. 

Cobbett1  considers  the  pseudodiphtheria  bacillus  as  perfectly  innocu- 
ous to  man,  but  that  the  relation  between  the  pseudodiphtheria  and 
the  diphtheria  bacillus  remains  undecided.  He  did  not  meet  with  bacilli 
of  low  virulence.  He  found  a  few  non-virulent  and  the  others  were  all 
highly  virulent.  He  thinks  that  the  reason  why  the  pseudodiphtheria 
bacilli  appear  so  infrequently  during  the  acute  stage  is  that  they  are 
overlooked  then  because  one  discovers  the  virulent  bacilli  so  easily 
and  does  not  trouble  to  look  any  more,  and  they  are  found  more  easily 
later  because  the  diphtheria  bacilli  are  disappearing  and  are  hard  to 
find;  consequently  a  long  and  careful  search  is  made,  and  the  pseudo- 
diphtheria  bacilli  are  seen  for  the  first  time. 

All  of  this  work  (including  the  reports  of  observers  not  mentioned 
in  this  paper)  in  regard  to  the  relationship  of  the  different  diphtheria- 
like  bacilli  to  the  true  diphtheria  bacillus  may  be  summed  up  and 
tabulated  as  follows: 

Statements  in  favor  of  the  belief  that  one  form  Statements  opposed  to  this  belief, 

may  be  changed  readily  into  another. 

1.  The  morphological  and  cultural  char-         The  morphological  and  cultural   charac- 

acteristics    of     all    diphtheria-like  teristics  of  varieties  have  many  points 

organisms   from   pseudo    to  typical  of  difference, 

virulent  forms  have  some  points  of 
resemblance. 

2.  Diphtheria     bacilli      possess     many        Intermediate  grades  of  virulence  are  rare. 

grades  of  virulence  from  the  fully 
virulent  to  the  non-virulent. 

3.  Non-virulent  bacilli,  both  typical  and        There  are  other    reasons    than   that  of 

non-typical,  have  been  found  more  change   of    one    form    to    another    to 

frequently  in  the  convalescing  stage  account  for  this, 
of  diphtheria  than  in  the  acute  stage. 

4.  Non-virulent,  atypical    bacilli    have  Virulent  diphtheria  bacilli  have  also  been 

been  the  only  diphtheria-like  organ-  frequently  found, 

isms  found  in  light  anginas. 

5.  A  sequence  of  forms  in  the  course  of        The   observation  is  correct   only  for  the 

diphtheria  and  in  successive  genera-  forms  in   the  original   mixed   cultures 

tions  of  pure  cultures,  from  granular  and   is  due  to   the  effect  of  the  other 

through  barred  to  solid  forms,  and  bacteria  on  the  development  of  the  diph- 

the  converse,  has  been  observed.  theria  bacilli  or  because  both  varieties 

were  present  at  the  start. 

i  Journal  of  Hygiene,  1901. 


202 


BACTERIA  PATHOGENIC  TO  MAN 


Statements  in  favor  of  the  belief  that  one  form 
may  be  changed  readily  into  another. 

6.  Solid  forms,  approaching  the  atypical 

non-virulent  forms,  have  been  found 
to  be  specifically  virulent. 

7.  The  virulence  of  the  diphtheria  bacil- 

lus has  been  decreased  artificially 
with  a  change  in  form  and  cultural 
characters,  and  slightly  virulent 
diphtheria  bacilli  have  been  made 
more  virulent. 

8.  Non-virulent    atypical   bacilli    have, 

in  a  few  hands,  been  changed  to 
typical,  specifically  virulent  diph- 
theria bacilli. 


9.  Virulent  typical  diphtheria  bacilli 
have  been  apparently  changed  to 
solidly  staining,  non-virulent,  diph- 
theria-like bacilli. 


Statements  opposed  to  this  belief. 

Among  large  numbers  of  virulent  diph- 
theria no  cultures  have  been  found 
which  developed  only  solid  varieties. 

Artificial  decrease  of  virulence  of  the 
diphtheria  bacillus  has  not  been  accom- 
plished easily,  neither  have  slightly 
virulent  bacilli  been  made  highly  viru- 
lent. 

Non-virulent  atypical  bacilli  experi- 
mented upon  by  most  observers  have 
retained  their  characteristics  on  various 
artificial  culture  media  under  different 
conditions  and  in  passage  through 
animals. 

Virulent  diphtheria  bacilli  usually  retain 
their  characteristics  on  artificial  culture 
media  under  different  conditions. 


The  central  idea  in  the  statements  of  those  who  believe  that  diph- 
theria-like bacilli  are  simply  transitory  variations  of  the  species  bacillus 
diphtheria  is  that  both  the  diphtheria  bacillus  and  those  bacilli  which 
resemble  them  have  many  unstable  properties,  their  form,  their  cul- 
tural characteristics,  their  pathogenicity  all  varying  within  a  wide 
limit,  so  that  one  form  may  assume  readily  the  properties  of  another 
form. 

The  separatists,  on  the  other  hand,  have  found  that  certain  forms 
possess  such  stable  properties  that  one  is  not  converted  into  another, 
and  hence  they  regard  them  as  distinct  species. 

In  order  to  make  a  thorough  test  of  this  whole  matter  Dr.  A.  W. 
Williams,  of  the  Research  Laboratory,  undertook  a  careful  investigation 
of  the  subject. 

An  outline  of  the  work  attempted  shows  the  thoroughness  of  the 
tests : 

1.  A  study  of  the  diphtheria  and  diphtheria-like  bacilli  found  in  a 
series  of  clinically  typical  diphtherias  at  the  Hospital  for  Contagious 
Diseases. 

(a)  Serial  smears  of  cultures  directly  from  throats  and  noses. 
(6)  Pure  cultures  isolated  from  these  cultures. 

2.  A  study  of  the  diphtheria  and  diphtheria-like  bacilli  found   in 
healthy  and  diseased  throats  in  a  town  during  an  epidemic  of  diphtheria. 

(a)  Smears  of  cultures  directly  from  throats. 
(6)  Pure  cultures  isolated  from  these  cultures. 

3.  A  study  of  diphtheria  and  diphtheria-like  bacilli  found  in  sore 
throats  during  an  epidemic  of  diphtheria  at  a  home  for  destitute  chil- 
dren. 

(a)  Pure  cultures. 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     203 

4.  A  study  of  pure  cultures  of  diphtheria  and  diphtheria-like  bacilli 
from  sources  other  than  those  given  above. 

(a)  On  various  artificial  culture  media  grown  under  various  con- 
ditions. 

(6)  In  living  tissues  of  guinea-pigs,  white  rats,  and  goldfinches. 

(c)  In  symbiosis  with  other  bacteria. 

The  conclusions  reached  were  as  follows:  Though  some  cultures 
change  on  some  of  the  media,  each  changes  in  its  own  way,  and  each 
culture  still  has  its  distinct  individuality.  After  many  culture  genera- 
tions, especially  when  transplanted  at  short  intervals,  the  different 
varieties  tend  to  approach  each  other  or  rather  to  run  in  lines  parallel 
with  a  common  norm,  which  seems  to  be  a  medium-sized,  non-seg- 
mented bacillus  producing  granules  in  early  cultures  on  serum  and 
growing  well  on  all  of  the  ordinary  culture  media.  The  non-virulent 
morphologically  typical  bacilli  must  be  classed  with  the  virulent  varieties 
as  one  species,  though  there  is  little  doubt  that  more  minute  study 
would  show  distinct  species  in  this  group.  The  atypical  pseudo 
forms,  however,  which  show  no  tendency  to  approach  the  norm  of 
the  typical  forms,  must  be  classed  as  distinct  species.  All  of  the 
pseudo  and  non-virulent  morphologically  typical  varieties  when  inocu- 
lated into  the  peritoneum  of  guinea-pigs  in  immense  doses  cause 
death.  Attempts  have  been  made  to  give  more  virulence  to  some  of 
these  varieties  by  successive  peritoneal  inoculations,  but  in  no  instance 
has  any  increase  of  virulence  or  decided  change  in  morphological  or 
cultural  characteristics  been  noted.  Two  of  the  non-virulent,  mor- 
phologically typical  varieties  have  also  been  grown  in  symbiosis  with 
virulent  streptococci  in  broth  for  ninety  culture  generations  trans- 
planted every  three  to  four  days,  but  when  separated  no  change  in 
virulence  or  other  characteristics  was  noted.  Two  other  varieties  of 
non-virulent  morphologically  typical  bacilli  have  been  inoculated  into 
goldfinches  with  no  result.  In  large  doses  they  appear  to  be  perfectly 
innocuous  to  these  birds  as  well  as  do  four  varieties  of  pseudobacilli, 
contrary  to  the  results  of  Richmond  and  Salter. 

Since  there  are  so  many  different  forms  or  varieties  of  diphtheria-like 
bacilli,  it  is  quite  possible  that  some  of  them  are  derived  from  strains  of 
the  diphtheria  bacillus  and  that  under  certain  conditions  they  readily 
regain  its  characteristics.  This  seems  to  be  the  only  way  to  explain  the 
apparent  discrepancies  in  the  results  obtained  by  different  observers. 
Such  closely  related  varieties,  however,  do  not  appear  to  be  common  in 
New  York  City  at  the  present  time.  So  we  may  safely  say  that  in  this 
region  at  least*  non-virulent  diphtheria-like  organisms  retain  their  char- 
acteristics under  various  artificial  and  natural  conditions,  and  that  they 
may  be  regarded  from  a  public  health  standpoint  as  harmless.  These 
studies  seem  to  demonstrate  that  the  morphologically  typical  diphtheria 
bacillus  is  a  distinct  species  from  the  atypical  diphtheria-like  bacilli  and 
so-called  pseudo  forms,  and  that  it  has  many  true  morphological  varieties 
or  sub-species  which,  while  showing  transitory  ontogenic  variations 
due  to  change  in  environment  and  life  habit,  have  more  or  less  per- 


204  BACTERIA  PATHOGENIC  TO  MAN 

sistent  phylogenic  characteristics  which  reappear  when  the  organism 
is  placed  in  a  previous  environment. 

Transmission  of  Diphtheria. — The  possibility  of  the  transmission  of 
diphtheria  from  animals  to  man  cannot  be  disputed ;  we  have  met  with 
one  instance  where  a  cat  had  malignant  diphtheria,  and  many  other 
animals  can  be  infected,  but  there  are  no  authentic  cases  of  such  trans- 
mission on  record.  So-called  diphtheritic  disease  in  animals  and  birds 
is  usually  due  to  other  micro-organisms  than  the  diphtheria  bacilli. 

Let  us  consider  some  of  the  means  by  which  the  disease  may  be  com- 
municated. In  actual  experiment  the  bacilli  have  been  observed  to 
remain  virulent  in  bits  of  dried  membrane  for  twenty  weeks.  Dried 
on  silk  threads  Abel  reports  that  they  may  sometimes  live  one  hundred 
and  seventy-two  days,  and  upon  a  child's  plaything  which  had  been 
kept  in  a  dark  place  they  lived  for  five  months.  The  virulent  bacilli 
have  been  found  on  soiled  bedding  or  clothing  of  a  diphtheria  patient, 
or  drinking-cups,  candy,  shoes,  hair,  slate-pencils,  etc.  Besides  these 
sources  of  infection  by  which  the  disease  may  be  indirectly  transmitted, 
virulent  bacilli  may  be  directly  received  from  the  pseudomembrane, 
exudate,  or  discharges  of  diphtheria  patients;  from  the  secretions  of  the 
nose  and  throat  of  convalescent  cases  of  diphtheria  in  which  the  virulent 
bacilli  persists;  and  from  the  healthy  throats  of  individuals  who  acquired 
the  bacilli  from  being  in  contact  with  others  having  virulent  germs  on 
their  persons  or  clothing.  In  such  cases  the  bacilli  may  sometimes 
live  and  develop  for  days  or  weeks  in  the  throat  without  causing  any 
lesion.  When  we  consider  that  it  is  only  the  severe  types  of  diphtheria 
that  remain  isolated  during  their  actual  illness,  the  wonder  is  not  that 
so  many,  but  that  so  few,  persons  contract  the  disease.  It  indicates 
that  very  frequently  virulent  bacilli  are  received  into  the  mouth,  and 
then  either  find  no  condition  there  suitable  for  their  growth  or  are 
swept  away  by  food  or  drink  before  they  could  effect  a  lodgement. 

Susceptibility  to  and  Immunity  against  Diphtheria. — An  individual 
susceptibility,  both  general  and  local,  to  diphtheria,  as  in  all  infectious 
diseases,  is  necessary  to  contract  the  disease.  Age  has  long  been  recog- 
nized to  be  an  important  factor  in  diphtheria.  Children  within  the 
first  six  months  of  life  are  but  little  susceptible,  the  greatest  degree  of 
susceptibility  being  between  the  third  and  the  tenth  year,  while  adults 
are  almost  immune. 

As  the  result  of  animal  experiments,  it  is  now  known  that  an  artificial 
immunity  against  diphtheria  can  be  produced,  at  least  for  a  consider- 
able length  of  time,  by  the  development  of  substances  directly  antidotal 
to  the  diphtheria  toxin.  By  the  inoculation  of  virulent  or  somewhat 
attenuated  cultures  or  of  diphtheria  toxin,  Fraenkel,  Behring,  Wernicke, 
Aronson,  Roux,  and  since  then  many  others,  have  succeeded  in  immun- 
izing animals;  but  the  most  important  and  valuable  results  are  those 
which  have  been  obtained  by  Behring,  in  conjunction  with  others, 
who  showed  that  the  blood  of  immune  animals  contains  a  substance 
which  neutralizes  the  diphtheria  toxin.  The  blood  serum  of  persons 
who  have  recovered  from  diphtheria  has  been  found  also  to  possess 


THE  HACILLUS  AXD  THE  HAC'I 7-.  ilIOLOGY  OF  DIPHTHERIA     205 

this  protective  property,  which  it  acquires  about  a  week  after  the  begin- 
ning of  the  disease,  and  loses  again  in  a  few  weeks  or  months.  More- 
over, the  blood  serum  of  many  individuals,  usually  adults,  who  have 
never  had  diphtheria  often  has  a  slight  general  antitoxic  property. 

Antitoxic  Serum. — The  knowledge  derived  from  these  remarkable 
investigations  into  the  protective  powers  of  the  blood  serum  of  immu- 
nized animals  has  been  employed  with  the  most  brilliant  results  for  the 
prevention  and  early  treatment  of  diphtheria  in  man.  The  discovery 
of  the  method  of  the  production  of  antitoxic  serum  or  antitoxin  in 
animals,  and  its  practical  application  to  the  treatment  and  cure  of 
diphtheria,  has  been  shared  by  many  experimenters,  at  first  chiefly  in 
Germany  and  France,  and  later  in  this  country. 

Results  of  the  Antitoxin  Treatment  of  Diphtheria. — The  conclusions 
arrived  at  by  Biggs  and  Guerard,  after  a  review  of  all  the  statistics  and 
opinions  published  since  the  beginning  of  the  antitoxin  treatment  in 
1892,  were  as  follows: 

"It  matters  not  from  what  point  of  view  the  subject  is  regarded,  if 
the  evidence  now  at  hand  is  properly  weighed,  but  one  conclusion  is  or 
can  be  reached — whether  we  consider  the  percentage  of  mortality  from 
diphtheria  and  croup  in  cities  as  a  whole,  or  in  hospitals,  or  in  private 
practice;  or  whether  we  take  the  absolute  mortality  for  all  the  cities  of 
Germany  whose  population  is  over  15,000,  and  all  the  cities  of  France 
whose  population  is  over  20,000;  or  the  absolute  mortality  for  New 
York  City,  or  for  the  great  hospitals  in  France,  Germany,  and  Austria; 
or  whether  we  consider  only  the  most  fatal  cases  of  diphtheria,  the 
laryngeal  and  operative  cases;  or  whether  we  study  the  question  with 
relation  to  the  day  of  the  disease  on  which  treatment  is  commenced, 
or  the  age  of  the  patient  treated;  it  matters  not  how  the  subject  is  re- 
garded or  how  it  is  turned  for  the  purpose  of  comparison  with  previous 
results,  the  conclusion  reached  is  always  the  same — namely,  there  has 
been  an  average  reduction  of  mortality  from  the  use  of  antitoxin  in  the 
treatment  of  diphtheria  of  not  less  than  50  per  cent.,  and  under  the  most 
favorable  conditions  a  reduction  to  one-quarter,  or  even  less,  of  the 
previous  death  rate.  This  has  occurred  not  in  one  city  at  one  particular 
time,  but  in  many  cities,  in  different  countries,  at  different  seasons  of 
the  year,  and  always  in  conjunction  with  the  introduction  of  antitoxin 
serum  and  proportionate  to  the  extent  of  its  use."  Except  where  im- 
munization has  been  practical  on  a  large  scale  no  reduction  in  the 
number  of  cases  of  diphtheria  has  been  evident. 

Production  of  Diphtheria  Antitoxin  for  Therapeutic  Purposes. — As  a 
result  of  the  work  of  years  in  the  laboratories  of  the  Health  Depart- 
ment of  New  York  City,  the  following  may  be  laid  down  as  a  practical 
method: 

A  strong  diphtheria  toxin  should  be  obtained  by  taking  a  very 
virulent  culture  and  growing  it  in  broth  under  the  conditions  described 
on  page  194.  The  culture,  after  a  week's  growth,  is  removed,  and 
having  been  tested  for  purity  by  microscopic  and  culture  tests  is  ren- 
dered sterile  by  the  addition  of  10  per  cent,  of  a  5  per  cent,  solution 


206  BACTERIA  PATHOGENIC  TO  MAN 

of  carbolic  acid.  After  forty-eight  hours  the  dead  bacilli  have  settled 
on  the  bottom  of  the  jar  and  the  clear  fluid  above  is  syphoned  off  or  it 
is  filtered  through  ordinary  sterile  filter  paper  and  stored  in  full  bot- 
tles in  a  cold  place  until  needed.  Its  strength  is  then  tested  by  giving 
a  series  of  guinea-pigs  carefully  measured  amounts.  Less  than  0.01 
c.c.,  when  injected  hypodermically,  should  kill  a  250-gram  guinea- 

pig- 

The  horses  used  should  be  young,  vigorous,  of  fair  size,  and  abso- 
lutely healthy.  Vicious  habits,  such  as  kicking,  etc.,  make  no  differ- 
ence, of  course,  except  to  those  who  handle  the  animals.  The  horses 
are  severally  injected  with  an  amount  of  toxin  sufficient  to  kill  five 
thousand  guinea-pigs  of  250  grams'  weight  (about  20  c.c.  of  strong 
toxin).  After  from  three  to  five  days,  so  soon  as  the  fever  reaction 
has  subsided,  a  second  subcutaneous  injection  of  a  slightly  larger  dose 
is  given.  With  the  first  three  injections  of  toxin  10,000  units  of 
antitoxin  are  given.  If  antitoxin  is  not  mixed  with  the  first  doses 
of  toxin  only  one-tenth  of  the  doses  advised  is  to  be  given.  At 
intervals  of  from  five  to  eight  days  increasing  injections  of  pure 
toxin  are  made,  until  at  the  end  of  two  months  from  ten  to  twenty 
times  the  original  amount  is  given.  *  There  is  absolutely  no  way  of 
judging  which  horses  will  produce  the  highest  grades  of  antitoxin. 
Very  roughly,  those  horses  which  are  extremely  sensitive  and  those 
which  react  hardly  at  all  are  the  poorest,  but  even  here  there  are  excep- 
tions. The  only  way,  therefore,  is  at  the  end  of  six  weeks  or  two  months 
to  bleed  the  horses  and  test  their  serum.  If  only  high-grade  serum  is 
wanted  all  horses  that  give  less  than  150  units  per  c.c.  are  discarded. 
If  moderate  grades  only  are  desired,  all  that  yield  100  units  may  be 
retained.  The  retained  horses  receive  steadily  increasing  doses,  the 
rapidity  of  the  increase  and  the  interval  of  time  between  the  doses 
(three  days  to  one  week)  depending  somewhat  on  the  reaction  follow- 
ing the  injection,  an  elevation  of  temperature  of  more  than  3°  F.  being- 
undesirable.  At  the  end  of  three  months  the  antitoxic  serum  of  all  the 
horses  should  contain  over  300  units,  and  in  about  10  per  cent,  as  much 
as  800  units  in  each  cubic  centimetre.  Very  few  horses  ever  give  above 
1000  units,  and  none  so  far  has  given  as  much  as  2000  units  per  c.c. 
The  very  best  horses  if  pushed  to  their  limit  continue  to  furnish  blood 
containing  the  maximum  amount  of  antitoxin  for  several  months,  and 
then,  in  spite  of  increasing  injections  of  toxin,  begin  to  furnish  blood  of 
gradually  decreasing  strength.  If  every  nine  months  an  interval  of  three 
months'  freedom  from  inoculations  is  given,  the  best  horses  furnish  high- 
grade  serum  during  their  periods  of  treatment  for  from  two  to  four  years. 

In  order  to  obtain  the  serum  the  blood  is  withdrawn  from  the  jugular 
vein  by  means  of  a  sharp-pointed  cannula,  which  is  plunged  through 
the  vein  wall,  a  slit  having  been  made  in  the  skin.  The  blood  is  carried 
by  a  sterile  rubber  tube  into  large  Erlenmeyer  flasks  and  allowed  to 
clot,  the  flasks,  however,  being  placed  in  a  slanting  position  before 
clotting  has  commenced.  The  serum  is  drawn  off  after  four  days  by 
means  of  sterile  glass  and  rubber  tubing,  and  is  stored  in  large  flasks. 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     207 

From  this,  as  needed,  small  phials  are  filled.  The  phials  and  their 
stoppers,  as  indeed  all  the  utensils  used  for  holding  the  serum,  must  be 
absolutely  sterile,  and  every  possible  precaution  must  be  taken  to  avoid 
contamination  of  the  serum.  An  antiseptic  may  be  added  to  the  serum 
as  a  preservative,  but  it  is  not  necessary  except  when  the  serum  is  to 
be  sent  to  great  distances,  where  it  cannot  be  kept  under  supervision. 

Kept  from  access  of  air  and  light  and  in  a  cold  place  it  is  fairly  stable, 
deteriorating  not  more  than  40  per  cent.,  and  often  much  less',  within 
a  year.  Diphtheria  antitoxin,  when  stored  in  phials  and  kept  under 
the  above  conditions,  contains  within  10  per  cent,  of  its  original  strength 
for  at  least  two  months;  after  that  it  can  be  used  by  allowing  for  a 
maximum  deterioration  of  5  per  cent,  for  each  month.  The  antitoxin 
in  old  serum  is  just  the  same  as  in  that  freshly  bottled,  only  there  is 
less  of  it.  Almost  all  producers  put  more  units  in  the  phials  than  the 
label  calls  for  so  as  to  allow  for  the  gradual  loss  of  strength. 

The  nature  of  diphtheria  antitoxin  has  until  recently  been  known 
almost  wholly  from  its  physiological  properties.  Experiments  have 
seemed  to  show  that  it  was  either  closely  bound  to  the  serum  globulins 
or  was  itself  a  substance  of  proteid  nature  closely  allied  to  serum 
globulin.  Mr.  J.  P.  Atkinson,  when  assistant  chemist  in  the  laboratory, 
found  that  antitoxic  and  normal  horse  serum  react  similarly  toward 
MgSO4,  in  that  the  globulin  is  precipitated  completely  from  the 
other  constituents  of  the  serum.  In  the  case  of  antitoxic  serum  the 
globulin  precipitate  carries  with  it  all  of  the  antitoxic  power  of  the 
serum,  leaving  the  filtrate  without  any  neutralizing  power  against 
the  diphtheria  toxin.  When  watery  solutions  of  this  globulin  are 
saturated  with  NaCl  a  precipitate  occurs.  When  the  solution  is  heated 
a  series  of  further  precipitates  take  place,  as  follows:  Cloudiness  appears 
at  40°,  49°,  57°,  and  67°  C.;  complete  precipitation  occurs  at  45°,  54°, 
62°,  and  72°  C.  Each  of  these  precipitates  has  antitoxic  properties, 
and  the  total  quantities  contain  all  the  original  antitoxin  except  ome 
5  per  cent.,  which  is  evidently  destroyed  by  the  higher  temperatures 
required  for  the  last  two  precipitates.  After  the  last  precipitate  the 
solution  is  free  of  globulin  and  also  of  all  antitoxic  properties. 

A  further  fact  developed  by  Atkinson  is  that  the  globulins  increase 
markedly  in  the  serum  of  horses  as  the  antitoxin  strength  increases. 
It  seems,  therefore,  from  the  above  that  diphtheria  antitoxin  has  the 
characteristics  of  the  serum  globulins.  Antitoxin  is  destroyed  by  pro- 
longed moderate  heat  (60°  C.)  and  by  short  exposure  to  higher  tem- 
peratures (95°  to  100°  C.).  It  is  less  sensitive  than  diphtheria  toxin. 

Diphtheria  antitoxin  has  the  power  of  neutralizing  diphtheria  toxin, 
so  that  when  a  certain  amount  is  injected  into  an  animal  before  or 
together  with  the  toxin  it  overcomes  its  poisonous  action.  The  facts 
in  favor  of  a  direct  action  of  antitoxins  upon  their  corresponding  toxins 
have  recently  been  briefly  summarized  by  Cobbett  as  follows: 

1.  Certain  reactions  j^ave  been  observed  to  take  place  between  these 
substances  outside  the  animal  body*  (venom,  ricin,  crotin,  tetanus  toxin, 
diphtheria  toxin,  and  their  corresponding  antitoxins). 


208  BACTERIA  PATHOGENIC  TO  MAN 

2.  Various   attempts   to   separate   the   toxins    and    antitoxins   from 
neutral  mixtures  have  been  failures.     Partial  successes  have,  at  least 
in  some  instances,  been  shown  to  depend  upon  the  fact  that  insufficient 
time  for  their  complete  union  was  allowed,  separation  being  no  longer 
possible  if  this  were  granted. 

3.  The  accuracy  of  the  titration  of  toxins  and  antitoxins  to  within 
1  per  cent,  of  error. 

4.  Neutralization  takes  place  according  to  the  law  of  multiple  pro- 
portions, i.  e.,  to  save  an  animal  from  1000  fatal  doses  of  diphtheria 
toxin  requires  little  more  than  a  hundred  times  as  much  antitoxin  as 
is  required  for  ten  fatal  doses,  the  resistance  of  the  animal  itself  account- 
ing for  the  difference. 

5.  The  fact  that  the  potency  of  antitoxin  is  greatly  increased  if  it  is 
allowed  to  come  in  contact  with  the  toxin  outside  the  animal  body; 
and  is  increased  still  further  if  allowed  to  remain  for  sufficient  time  in 
contact  with  the  toxin  at  a  suitable  temperature.     The    union  takes 
place  more  quickly  at  a  warm  than  at  a  cold  temperature. 

The  facts  now  known,  therefore,  indicate  that  the  antitoxins  of 
tetanus  and  diphtheria,  of  snake-poison,  of  ricin,  etc.,  enter  into  direct 
chemical  combination  with  their  respective  toxins.  Many  points, 
however,  are  still  far  from  clear  as  to  the  manner  in  which  both  toxins 
and  antitoxins  act. 

Testing  of  Antitoxin. — This  power,  possessed  by  a  definite  quan- 
tity of  antitoxin  to  neutralize  a  certain  amount  of  toxin,  is  utilized  in 
testing  antitoxin.  Guinea-pigs  of  about  250  grams'  weight  are  sub- 
cutaneously  injected  with  one  hundred  or  with  ten  fatal  doses  of  a 
standardized  toxin,  which  have  been  previously  mixed  with  an  amount 
of  antitoxin  believed  to  be  sufficient  to  protect  from  the  toxin.  If  the 
guinea-pig  lives  four  days,  but  dies  soon  after,  the  amount  of  anti- 
toxin added  to  the  one  hundred  fatal  doses  of  toxin  was  just  1  unit.  If 
the  guinea-pig  dies  earlier,  less  than  1  unit  was  added. 

Use  of  Antitoxin  in  Treatment  and  Immunization. — The  antitoxin 
in  the  higher  grades  is  identical  with  that  in  the  lower  grades;  there  is 
simply  more  of  it  in  each  drop  of  the  serum.  In  treatment,  however, 
for  the  same  amount  of  antitoxin  we  have  to  inject  less  blood  serum 
with  the  higher  grades,  and,  therefore,  have  somewhat  less  danger  of 
rashes  and  other  deleterious  results.  The  amount  of  antitoxin  required 
for  immunization  is  300  units  for  an  infant,  500  for  an  adult,  and  propor- 
tionately for  those  between  these  extremes.  After  the  observation  of 
the  use  of  antitoxin  in  the  immunization  of  several  thousand  cases,  I 
have  absolute  belief  in  its  power  to  prevent  an  outbreak  of  diphtheria 
for  at  least  two  weeks,  and  also  of  its  almost  complete  harmlessness 
in  the  small  doses  required.  If  it  is  desired  to  prolong  the  immunity 
the  antitoxin  injection  is  repeated  every  two  weeks.  For  treatment, 
mild  cases  should  be  given  1500  units,  moderate  cases  2000  to  4000 
units,  and  severe  cases  5000  units.  Where  n^LJmprovement  follows 
in  twelve  hours  the  dose  should  be  repeated.  Intravenous  injections  give 
most  rapid  effect.  Antitoxin  is  not  absorbed  when  given  by  the  mouth. 


Till-:  BACILLUS  AXD  THE  BACTERIOLOGY  OF  DIPHTHERIA     209 

No  deleterious  effects  are  to  l>e  feared  except  a  rash,  with  some  rise 
of  temperature,  in  about  20  per  cent,  of  the  cases.  In  about  1  per 
cent,  of  the  cases  swelling  and  tenderness  of  one  or  more  joints  occur. 
Except  in  septic  cases  no  permanent  disability  follows. 

There  are  on  record  some  five  or  six  cases  where  following  an  injec- 
tion in  a  case  of  diphtheria  sudden  death  has  followed.  The  result  is 
probably  due  to  the  excitement  caused  by  the  operation  rather  than  to 
the  serum. 

With  the  serum  from  some  horses  the  rashes  are  very  infrequent, 
while  with  that  from  others  they  occur  more  often.  The  same  horse 
will  at  one  time  furnish  a  serum  which  produces  no  rashes  and  at 
another  one  which  gives  a  great  number.  No  way  has  yet  been  found 
to  eliminate  them  entirely.  Filtering  and  moderate  heating  produce 
little  effect.  Standing  for  some  months  causes  a  precipitate  to  occur, 
and  the  clear  serum  seems  somewhat  less  liable  to  produce  rashes 
than  when  it  was  fresh. 

Use  of  a  Serum  to  Eradicate  Diphtheria  Bacilli  from  Convalescents 
and  Healthy  Persons. — A  great  difficulty  in  combating  diphtheria  is 
this :  that  in  healthy  children,  but  especially  in  diphtheria  convalescents, 
despite  the  use  of  antitoxin,  the  diphtheria  bacilli  often  remain  in  the 
nasopharynx  for  a  very  long  time.  This  is  extremely  annoying,  because 
a  child  so  affected  cannot  be  sent  to  school  until  all  diphtheria  bacilli 
have  disappeared  from  the  nasopharynx.  Wassermann  has  done  as 
follows:  A  strongly  agglutinating,  multipartial  diphtheria  serura  is 
evaporated  to  dryness  in  vacuo,  mixed  with  sugar  of  milk,  pulverized, 
and  pressed  into  tablets.  These  tablets  when  dissolved  in  the  mouth 
cause  the  fluids  in  the  mouth  to  become  strongly  agglutinating.  The 
question  was,  and  is,  whether  this  process  of  agglutination  will  help  us 
to  get  rid  of  the  diphtheria  bacilli  from  the  nasopharynx  more  quickly 
and  surely  than  was  heretofore  possible.  His  clinical  experiments 
thus  far  made  speak  in  favor  of  the  employment  of  this  serum.  Whereas, 
it  is  no  rarity  for  diphtheria  bacilli  to  be  present  in  the  throats  of  con- 
valescents for  weeks,  he  found  that  in  the  cases  in  which  these  tablets 
have  been  used  the  bacilli  disappeared  within  a  few  days. 

His  method  is  this:  A  tablet  is  allowed  to  dissolve  in  the  mouth 
about  every  two  hours,  and  then,  after  fifteen  minutes,  the  child's 
nasopharynx  is  rinsed  out  with  an  indifferent  fluid  in  the  form  of  a 
spray  or  gargle.  He  conceives  the  action  to  be  such  that,  whereas  when 
this  serum  is  not  employed  the  diphtheria  bacilli  are  scattered  diffusely 
throughout  the  nasopharynx,  under  the  influence  of  this  serum  they 
are  agglutinated  or  clumped  together.  The  diphtheria  bacilli  are 
massed  together  more  or  less  by  the  serum,  and  these  clumps  are  then 
removed  by  the  subsequent  rinsing.  In  this  way  they  are  so  much 
decreased  in  amount  that  the  natural  power  of  the  organism  is  able 
much  more  quickly  to  make  away  with  those  remaining.  He  has  hopes 
that  this  new  diphtheria  serum  will  be  destined  to  be  of  great  service, 
especially  in  making  prophylaxis  easier  and  in  making  it  possible  to 
send  the  diphtheria  convalescents  to  school  earlier  than  heretofore. 

14 


210 


BACTERIA  PATHOGENIC  TO  MAN 


My  own  experience  with  this  serum  have  been  few  and  unsatisfac- 
tory. 

Development  of  Agglutinins  for  Diphtheria  Bacilli. — By  the  injec- 
tions of  the  bodies  of  diphtheria  bacilli  into  animals  agglutinins  have 
been  developed  in  sufficient  amount  to  act  in  1 : 5000  dilutions  of  the 
serum.  The  serum  produced  from  diphtheria  bacilli  does  not  agglu- 
tinate pseudodiphtheria  bacilli  in  high  dilutions.  The  serum  of  patients 
convalescent  from  diphtheria  has,  as  a  rule,  little  agglutinating  power. 
This  test  is  not  used  in  diagnosis. 

Persistence  of  Antitoxin  in  the  Blood. — When  injections  of  toxin  are 
stopped  in  a  horse  the  antitoxin  is  slowly  eliminated,  so  that  there 
is  a  loss  of  about  20  per  cent,  a  week.  In  from  three  to  five  months 
all  appreciable  antitoxin  has  been  eliminated. 

Technical  Points  upon  the  Testing  of  Diphtheria  Antitoxin  and  the  Rela- 
tions between  the  Toxicity  and  Neutralizing  Value  of  Diphtheria  Toxin.— 
During  the  earlier  investigations  the  filtered  or  sterilized  bouillon,  in 
which  the  diphtheria  bacillus  had  grown  and  produced  its  "toxin," 
was  supposed  to  require  for  its  neutralization  an  amount  of  antitoxin 
directly  proportional  to  its  toxicity  as  tested  in  guinea-pigs.  Thus, 
if  from  one  bouillon  culture  ten  fatal  doses  of  "toxin"  were  required 
to  neutralize  a  certain  quantity  of  antitoxin,  it  was  believed  that  ten 
fatal  doses  from  every  culture,  without  regard  to  the  way  in  which  it 
had  been  produced  or  preserved,  would  also  neutralize  the  same  amount 
of  antitoxin.  Upon  this  belief  was  founded  the  Behring-Ehrlich  defini- 
tion of  an  antitoxin  unit. 

The  results  of  tests  by  different  experimenters  with  the  same  anti- 
toxic serum,  but  with  different  diphtheria  toxins,  proved  this  opinion 
to  be  incorrect.  Ehrlich1  deserves  the  credit  for  first  clearly  perceiving 
and  publishing  this.  He  obtained  from  various  sources  twelve  toxins 
and  compared  their  neutralizing  value  upon  antitoxin;  these  tests  gave 
most  interesting  and  important  information.  The  results  in  four  toxins, 
which  are  representative  of  the  twelve,  are  as  shown  in  the  following 
table: 


Toxin 
specimen 
number 
of 
Ehrlich. 

Estimated 
"minimal" 
fatal  dose 
for250-gm. 
guinea- 
pigs. 

Smallest  number  of 
fatal  doses  of  toxic 
bouillon  required  to   ; 
kill  a  250-gm  guinea- 
pig  within  5  days, 
when  mixed  with  one 
antitoxin  unit. 
"  L  ,   Ehrlich." 

Fatal  doses  required 
to  "  completely 
neutralize  one  anti- 
toxin unit"  as  de- 
termined by  the 
health  of  the  guinea- 
pig  remaining  un- 
affected. 
"L0"  Ehrlich. 

L+-L0 

=  fatal 
doses. 

Data  upon  "toxin" 
specimen  given  by 
Ehrlich. 

4 
7 
9 

12 

0.009 
0.0165 
0.039 

0.0025 

39.4 
76.3 
123 

100 

33.4 
54.4 
108 

50 

6 
22 
15 

50 

Old,  deteriorated  from 
0.003  to  0.009 
Fresh  toxin,  preserved 
with  tricresol. 
A  number  of  fresh  cul- 
tures grown  at  37°  C. 
four  and  eight  days. 
Tested  immediately 
after  its  withdrawal. 

'  Die  Wertbemessung  des  Diphtherieheilserums  und  deren  theoretisehe  Grundlagen.  Klinisches 
Jahrbuch,  1897. 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     211 

From  the  facts  set  forth  in  the  table,  Ehrlich  believed  that  the  diph- 
theria bacilli  in  their  growth  produce  a  toxin  which,  so  long  as  it  remains 
chemically  unaltered,  has  a  definite  poisonous  strength  with  a  definite 
value  in  neutralizing  antitoxin.  This  neutralization  he  believed  to  be 
a  chemical  union,  in  which  two  hundred  fatal  doses  of  toxin  for  a  250 
grams'  weight  guinea-pig  combine  with  one  unit  of  antitoxin.  The 
toxin  is,  however,  an  unstable  compound,  and  begins  to  change  almost 
immediately  into  substances  which  are  not,  at  least  acutely,  poisonous, 
but  which  retain  their  full  power  to  neutralize  antitoxin.  These  sub- 
stances, according  to  Ehrlich,  fell  into  three  groups.  The  first  has 
more  affinity  for  combining  with  the  antitoxin  than  the  toxin  itself 
(protoxoids).  The  second  has  the  same  affinity  (syntoxoids).  The 
third  has  less  affinity  (epitoxoids).  The  development  of  Ehrlich's 
theories  of  the  chemical  nature  of  this  union  of  pure  and  modified 
toxin  with  antitoxin  is  described  on  page  165.  The  toxin  with 
its  haptophore  group  intact  but  with  its  toxophore  altered  is  the 
toxoid. 

According  to  him,  if  a  mixture  of  toxoids  and  toxin  is  added  to  anti- 
toxin, the  protoxoids  first  combine  with  the  antitoxin;  then  the  syn- 
toxoids and  the  toxin  combine  in  equal  proportions,  so  long  as  the 
supply  lasts,  with  the  amount  of  antitoxin  remaining,  or,  if  there  is  a 
surplus,  with  enough  to  satisfy  them;  finally,  if  any  antitoxin  remains, 
the  epitoxoids  unite  with  it. 

The  results  of  these  experiments  of  Atkinson  and  myself1  were  fully 
in  accord  with  those  published  by  Ehrlich  as  to  the  varying  neutral- 
izing value  of  a  minimal  fatal  dose  of  "toxin;"  they,  however,  also 
indicate  roughly  a  general  law  in  accordance  with  which  these  changes 
occur. 

The  neutralizing  value  of  a  fatal  dose  of  toxin  is  at  its  lowest  in  the 
culture  fluid  when  the  first  considerable  amounts  of  toxin  have  been 
produced.  After  a  short  period,  during  which  the  quantity  of  toxin 
in  the  fluid  is  increasing,  the  neutralizing  value  of  the  fatal  dose  begins 
to  increase,  at  first  rapidly,  then  more  slowly. 

While  the  culture  is  still  in  vigorous  growth  and  new  toxin  is  being 
produced,  the  neutralizing  value  of  the  fatal  dose  fluctuates  somewhat, 
but  with  a  generally  upward  tendency.  After  the  cessation  of  toxin 
production  the  neutralizing  value  of  the  fatal  dose  increases  steadily 
until  it  becomes  five  to  ten  times  its  original  amount. 

In  our  experiments  the  greatest  value  for  L+  was  126,  the  least  27. 
As  at  six  hours  L+  was  only  72  and  at  twenty-eight  hours  only  91,  we 
doubt  whether  L+  ever  reaches  above  150.2  When  we  seek  to  analyze 
the  above-described  process  we  find  certain  facts  which  seem  partly 
to  explain  it. 

In  the  fluid  holding  the  living  bacilli  we  have,  after  the  first    few 

1  Journal  of  Experimental  Medicine,  vol.  iii.,  No.  4. 

*  L+  =  fatal  doses  of  toxin  required  to  kill  a  guinea-pig  in  four  days  after  having  been  mixed 
>vith  one  unit  of  antitoxin. 
LO  =  fatal  doses  of  toxin  required  to  fully  neutralize  one  unit  of  antitoxin. 


212  BACTERIA  PATHOGENIC  TO  MAN 

hours  of  toxin  formation,  a  double  process  going  on — one  of  deteri- 
oration in  the  toxin  already  accumulated,  which  tends  to  increase 
the  neutralizing  value  of  the  fatal  dose ;  the  other  of  new  toxin  forma- 
tion, which  probably  tends  to  diminish  the  neutralizing  value.  The 
chemical  changes  produced  by  the  growth  of  the  bacilli  in  the  bouillon 
tend  to  aid  one  or  the  other  of  these  processes,  and  so  to  make,  from 
hour  to  hour,  slight  changes  in  the  value  of  the  fatal  dose.  Later,  with 
the  period  of  cessation  of  toxin  production,  the  gradual  deterioration  of 
the  toxicity  alone  continues,  and  the  fatal  dose  gradually  and  steadily 
increases  in  its  neutralizing  value. 

With  greater  information  Ehrlich  has  had  to  modify  greatly  the 
details  of  his  explanation  of  the  reason  of  the  variation  in  the  ratio 
between  toxicity  and  neutralizing  value  of  toxin.  He  now  accepts  the 
fact  that  diphtheria  culture  fluid  contains  at  least  two  toxins. 

Partial  Saturation  Method  of  Study. — Much  additional  information 
concerning  the  nature  of  toxin  has  been  gained  by  experimenting  with 
mixtures  of  toxin  and  antitoxin,  in  which  the  two  are  present  in  varying 
proportions.  This  is  the  "partial  saturation"  method  of  Ehrlich. 
Through  a  number  of  experiments  Ehrlich  obtained  information  which 
permitted  him  to  estimate  that  200  "binding  units"  are  represented 
in  the  amount  of  diphtheria  toxin  (hypothetically  pure)  which  is  exactly 
neutralized  by  one  antitoxin  unit.  If  the  entire  amount  of  antitoxin— 
i.  e.,  200/200,  is  added  to  the  amount  of  toxin  in  question,  complete 
neutralization  of  the  latter,  of  course,  occurs.  In  case  the  toxin  is  entirely 
pure,  199/200  of  the  antitoxin  unit  would  destroy  all  but  1/200  of  the 
initial  toxicity;  and  150/200,  or  100/200,  or  75/200,  etc.,  of  the  antitoxin 
when  added  would  permit  corresponding  degrees  of  toxicity  to  be 
demonstrated  through  animal  inoculations.-  It  was  found,  however, 
that  neutralization  according  to  this  simple  scale  did  not  take  place. 
The  results  were  complicated,  and  Ehrlich  has  found  it  convenient  to 
express  them  graphically  in  the  form  of  the  "toxin  spectrum."  For 
example,  let  199/200  of  the  antitoxin  unit  be  added  to  the  proper  amount 
of  the  toxin,  198/200  to  another  similar  amount,  197/200  to  another,  etc., 
down  to  150/200.  In  the  last  mixture,  50  out  of  the  200  binding  units 
which  the  toxin  possesses  are  free,  and  these  50,  rather  than  some  other 
50,  are  free  because  they  have  less  affinity  for  the  antitoxin  than  the  150 
units  which  were  bound.  It  has  been  found  that  those  units  which 
first  become  free  are  much  less  toxic  than  a  corresponding  amount  of 
the  original  toxin.  It  was  thought  that  they  might  have  lost  their  toxo- 
phore  groups — i.  e.,  that  they  were  toxoids;  and  because  of  their  weak 
affinity  for  antitoxin  they  were  called  epitoxoids.  It  was  found,  how- 
ever, that  they  possessed  a  rather  constant  though  low  degree  of  toxicity 
and  that  the  toxic  action  was  characteristic.  Injection  was  followed 
by  some  local  oedema,  then  by  a  long  incubation  period,  and  finally  by 
cachexia  and  paralysis.  On  account  of  this  characteristic  toxic  action 
and  the  long  incubation  period,  Ehrlich  has  concluded  that  the  so-called 
epitoxoid  is  in  reality  a  separate  toxin  secreted  by  the  diphtheria 
bacillus. 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     213 

Toxon. — This  he  now  designates  as  toxon  in  order  to  distinguish  it 
from  that  other  constituent  of  diphtheria  bouillon,  the  toxin,  which 
causes  the  acute  phenomena  of  diphtheria. 

Let  one  now  add  still  smaller  amounts  of  the  antitoxin  unit  to  the 
200  binding  units  of  the  toxin.  When  149/200  are  added  it  is  found  that 
a  certain  amount  of  true  toxin  remains  free,  and,  moreover,  is  free  in 
direct  proportion  to  the  amount  of  antitoxin  withheld.  Consequently 
when  but  50/200  antitoxin  unit  is  added  the  amount  of  free  toxin  corre- 
sponds to  100  binding  units.  If  true  toxin  only  remained  a  continua- 
tion of  the  experiment  would  show  toxin  equals  150.  It  could  then  be 
said  that  the  constitution  of  this  toxin  is :  toxin  150  and  toxon  50.  How- 
ever, it  may  be  found  that  as  49/200,  48/200,  etc.,  to  0/200  antitoxin 
unit  are  added,  no  increase  of  free  toxin  is  found,  although  the  antitoxin 
added  has  been  found.  Therefore,  the  50  toxin  binding  units  which 
have  the  greatest  affinity  for  antitoxin  are  non-toxic — i.  e.,  they  are 
toxoids,  and  since  they  have  the  maximum  affinity  for  antitoxin  they 
are  called  protoxoids. 

It  has  been  assumed  also  that  a  toxoid  may  exist  which  has  an  affinity 
for  antitoxin  exactly  equalling  that  which  toxin  possesses;  this  as  yet 
purely  hypothetical  constituent  bears  the  name  of  syntoxoid. 

Refinements  in  experimentation  show  that  even  the  true  toxin  is  not 
uniform  in  its  virulence  and  its  affinity  for  antitoxin.  Accordingly,  a 
prototoxin,  a  deuterotoxin,  and  a  tritotoxin  may  be  recognized  by  this 
same  partial  saturation  method.  For  example,  it  may  be  found  that 
when  a  portion  of  the  antitoxin  unit,  between  the  limits  of  149/200  and 
125  200,  is  withheld,  a  toxin  is  left  free  wrhich  is  less  virulent  than  that 
remaining  free  between  the  limits  of  124/200  and  100/200;  and  from  this 
point  on  the  new  unbound  toxin  may  be  still  more  virulent.  The  first 
would  be  tritotoxin,  the  second  deuterotoxin,  and  the  third  prototoxin. 

A  "spectrum"  having  been  \vorked  out  for  a  toxin  when  fresh,  an 
examination  made  some  time  later,  a  year  for  example,  may  show  many 
changes.  The  prototoxin  zone  and  portions  of  the  deuterotoxin  or 
tritotoxin  may  also  have  disappeared  because  of  toxoid  formation. 
These  changes  have  led  to  the  recognition  of  an  alpha  and  a  beta 
modification  of  the  toxin  portions.  The  alpha  modifications  of  all 
three  toxins  readily  become  toxoids.  Only  the  beta  modification  of 
the  deuterotoxin  remains  constant.  The  toxon  portion  also  remains 
relatively  intact. 

Summary. — To  summarize  Ehrlich's  views  as  to  the  nature  of  diph- 
theria toxin :  The  diphtheria  bacillus  secretes  two  toxins,  one  of  which, 
the  toxin,  causes  the  acute  phenomena  of  diphtheria  intoxication,  while 
the  other,  the  toxon,  causes  cachexia  and  paralysis  after  a  rather  long 
period  of  incubation.  The  non-toxic  toxin,  or  toxoid,  appears  as  the 
result  of  the  degeneration  of  the  toxophore  group  of  the  toxin,  the 
haptophore  group  remaining  intact.  The  toxin  may  be  separated  into 
three  divisions,  which  vary  in  their  affinity  for  antitoxin — prototoxin, 
deuterotoxin,  and  tritotoxin.  On  the  same  basis  there  are  three  toxoids 
—protoxoids,  syntoxoids,  and  epi toxoid  (the  toxon) — the  first  having 


214  BACTERIA  PATHOGENIC  TO  MAN 

the  greatest  affinity  for  antitoxin,  while  the  epitoxoid  has  the  least. 
The  toxins  are  divided  into  an  alpha  and  a  beta  portion,  depending  on 
the  ease  with  which  they  are  changed  into  toxoids.  All  of  these  sub- 
stances unite  with  tissue  cells  and  with  antitoxin  through  the  agency 
of  a  haptophore  group,  while  the  toxicity  depends  on  the  presence  of 
a  toxophore  group  in  the  toxin  or  toxon  molecule. 

Bordet  and  others  refuse  to  accept  these  complicated  conceptions 
of  Ehrlich  and  the  whole  matter  is  at  the  present  time  under  active 
discussion.  Thus  the  existence  or  non-existence  of  toxons  has  excited 
a  great  deal  of  discussion  among  investigators.  The  great  Swedish 
chemist,  Arrhenius,  has  recently  given  much  attention  to  toxins  and  is 
applying  the  principles  of  physical  chemistry  to  the  study  of  toxins 
and  antitoxins.  It  is  a  well-known  fact  that  some  chemical  substances 
when  in  solution  have  the  power  of  breaking  up  into  their  constituent 
parts;  thus  sodium  chloride  breaks  up  in  part  into  sodium  and  chlorine, 
as  sodium  or  chlorine  ions  or  electrolytes.  The  dissociated  sodium  and 
chlorine  may  then  enter  into  combination  with  any  other  suitable  sub- 
stance which  may  be  present.  Arrhenius  holds  that  this  is  the  case 
with  the  toxin-antitoxin  molecule,  that  it  may  to  a  certain  extent  again 
break  up  into  separate  toxin  and  antitoxin.  He  believes  that  this  dis- 
sociated toxin  is  the  substance  which  Ehrlich  has  been  calling  toxon. 
Madsen,  who  formerly  had  done  much  work  with  toxons,  has  now 
joined  with  Arrhenius  in  support  of  the  dissociation  theory.  In  spite  of 
their  reasoning  Ehrlich  and  his  followers  continue  to  uphold  the  toxon 
as  an  independent  toxic  substance.  Recent  investigations  throw  doubt 
on  both  explanations  as  being  at  all  final. 

Standardizing  of  Antitoxin  Testing. — Ehrlich  has  contributed  greatly 
to  uniformity  in  results  in  testing  antitoxin  by  calling  attention  to 
the  necessity  of  selecting  a  suitable  toxin  and  by  employing  and 
distributing  an  antitoxin  as  a  standard  to  test  toxins  by.  In  this  way 
smaller  testing  stations  can  make  their  results  correspond  with  those  of 
the  central  station.  The  United  States  Marine  Hospital  laboratories 
have  recently  begun  to  distribute  to  laboratories  in  the  United  States 
an  equally  carefully  standardized  serum. 

In  spite  of  the  great  variations  in  the  neutralizing  value  of  a  fatal 
dose  in  different  toxins  we  do  not  believe  that  even  before  adopting  the 
use  of  a  standard  serum  there  has  been  any  such  great  difference  in 
the  toxins  used  by  the  different  stations  for  testing  purposes.  Most 
laboratories  have  taken  the  culture  fluid  at  about  the  time  of  its  greatest 
toxicity,  and  the  neutralizing  value  of  a  fatal  dose  of  this  toxin  would 
seldom  vary  more  than  10  per  cent,  above  or  below  the  standard  now 
adopted  in  Germany  by  the  government  testing  station. 

Where  error  has  been  made  it  has  usually  been  by  taking  too  old 
culture  fluids,  which  would  cause  the  antitoxin  strength  of  samples 
tested  to  be  estimated  below  and  not  above  its  real  value.  Culture  8, 
which  is  used  not  only  by  the  New  York  Board  of  Health  Laboratory, 
but  by  many  other  laboratories  in  the  United  States  and  Europe,  for- 
tunately produces  on  the  sixth  to  eighth  day — the  time  at  which  the 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     215 

culture  is  usually  removed — a  toxin  which  usually  grades  Ehrlich's 
antitoxin  within  5  per  cent,  of  the  strength  given  by  him. 

We  believe  that  by  using  such  a  bacillus  we  can,  after  gaining  a  fuller 
knowledge  of  its  characteristics,  obtain  a  toxin  of  a  known  and  suitable 
neutralizing  value,  and  thus  always  correctly  standardize  an  antitoxic 
serum  in  case  the  present  stations  ceased  to  supply  a  testing  serum. 
This  is  certainly  true  for  the  bacillus  which  we  have  used  for  the 
past  ten  years.  A  preparation  of  a  carefully  tested  antitoxin  is  of 
immense  value  in  ensuring  a  uniform  standard  among  the  different 
testing  stations  and  in  allowing  of  comparison  between  them. 

The  old  definition  of  Behring  and  Ehrlich,  that  an  antitoxin  unit 
contains  the  amount  of  antitoxin  which  will  protect  the  life  of  a  guinea- 
pig  from  one  hundred  fatal  doses  of  toxin,  must  be  modified  so  as  to 
be  defined  as  that  amount  of  antitoxin  which  will  neutralize  one  hun- 
dred fatal  doses  of  a  toxin  similar  to  that  adopted  as  the  standard — 
namely,  one  having  approximately  the  characteristics  of  toxins  in  cul- 
tures at  the  height  of  their  toxicity. 

The  actual  test  of  an  antitoxin  serum  is,  therefore,  carried  out  as 
follows:  Six  guinea-pigs  are  injected  with  mixtures  of  toxin  and  anti- 
toxin. In  each  of  the  mixtures  there  is  100  times  the  amount  of  a  toxin 
such  as  just  described,  which  will  kill  250  grm.  of  guinea-pig  on  an 
average  in  ninety-six  hours.  In  each  of  the  mixtures  the  amount  of 
antitoxin  varies;  for  instance,  No.  1  would  contain  0.002  c.c.  serum; 
No.  2,  0.003  c.c.;  No.  3,  0.004  c.c.;  No.  4,  0.005  c.c.,  etc.  If  at  the  end 
of  the  fourth  day  Nos.  1,  2,  and  3  were  dead  and  Nos.  4,  5,  and  6 
were  alive  we  would  consider  the  serum  to  contain  200  units  of  anti- 
toxin for  each  cubic  centimetre.  When  we  mix  only  ten  fatal  doses 
of  toxin  with  one-tenth  of  the  amount  of  antitoxin  used  with  one  hun- 
dred fatal  doses  the  guinea-pig  must  remain  well.  The  mixed  toxin 
and  antitoxin  must  remain  together  for  fifteen  minutes  before  injecting. 

Relation  of  Bacteriology  to  Diagnosis. — We  believe  that  all  experienced 
clinicians  will  agree  that,  when  left  to  judge  solely  by  the  appearance 
and  symptoms  of  a  case,  there  are  certain  mild  exudative  inflammations 
of  the  throat  which  are  at  times  excited  by  diphtheria  bacilli  and  at 
times  by  other  bacteria. 

It  is  not  meant  to  imply  that  a  case  is  one  of  true  diphtheria 
simply  because  the  diphtheria  bacilli  are  present,  but  rather  that  the 
doubtful  cases  not  only  have  the  diphtheria  bacilli  in  the  exudate, 
but  are  capable  of  giving  true  characteristic  diphtheria  to  others, 
or  later  develop  it  characteristically  themselves;  and  that  those  in 
whose  throats  no  diphtheria  bacilli  exists  can  under  no  condition  give 
true  characteristic  diphtheria  to  others,  or  develop  it  themselves 
unless  they  receive  a  new  infection.  It  is,  indeed,  true,  as  a  rule, 
that  cases  presenting  the  appearance  of  ordinary  follicular  tonsillitis 
in  adults  are  not  due  to  the  diphtheria  bacillus.  It  is  also  true  that 
now  and  then  a  case  having  this  appearance  is  one  of  diphtheria,  and 
almost  every  physician  has  seen  such  cases  from  time  to  time  in  house- 
holds infected  with  diphtheria.  On  the  other  hand,  in  small  children 


216  BACTERIA  PATHOGENIC  TO  MAN 

mild  diphtheria  very  frequently  occurs  with  the  semblance  of  rather 
severe  ordinary  follicular  tonsillitis,  due  to  the  pyogenic  cocci,  and  in 
large  cities  where  diphtheria  is  prevalent  all  such  cases  must  be  watched 
as  being  more  or  less  suspicious.  As  showing  doubt  in  our  judg- 
ment, I  think  most  would  feel  that  if  in  any  case  exposure  to  diphtheria 
is  known  to  have  occurred,  even  a  slightly  suspicious  sore  throat  would 
be  regarded  as  probably  due  to  the  diphtheria  bacilli.  If,  on  the  other 
hand,  no  cases  of  diphtheria  have  been  known  to  exist  in  the  neighbor- 
hood, even  cases  of  a  more  suspicious  nature  would  probably  not  be 
regarded  as  diphtheria. 

Appearances  Characteristic  of  Diphtheria. — The  presence  of  irregular- 
shaped  patches  of  adherent  grayish  or  yellowish-gray  pseudomembrane 
or  some  other  portions  than  the  tonsils  is,  as  a  rule,  an  indication  of  the 
activity  of  the  diphtheria  bacilli.  Restricted  to  the  tonsils  alone  their 
presence  is  less  certain. 

Occasionally,  in  scarlatinal  angina  or  in  severe  phlegmonous  sore 
throats,  patches  of  exudate  may  appear  on  the  uvula  or  borders  of  the 
faucial  pillars,  and  still  the  case  may  not  be  due  to  the  diphtheria  bacilli; 
these  are,  however,  exceptional.  Thick,  grayish  pseudomembranes 
which  cover  large  portions  of  the  tonsils,  soft  palate,  and  nostrils  are 
almost  invariably  the  lesions  produced  by  diphtheria  bacilli. 

The  very  great  majority  of  cases  of  pseudomembranous  or  exudative 
laryngitis,  in  the  coast  cities  at  least,  whether  an  exudate  is  present  in 
the  pharynx  or  not,  are  due  to  the  diphtheria  bacilli.  Cases  in  which 
no  exudate  is  apparent  and  those  in  which  the  laryngeal  obstruction  is 
sudden  and  the  spasmodic  element  is  marked  are,  however,  frequently 
due  to  the  activity  of  other  bacteria.  Nearly  all  membranous  affec- 
tions of  the  nose  are  true  diphtheria.  When  the  membrane  is  limited 
to  the  nose  the  symptoms  are,  as  a  rule,  very  slight;  but  when  the  naso- 
pharynx is  involved  the  symptoms  are  usually  grave.  Ordinarily  a 
small  area  of  inflammation  indicates  a  slight  or  moderate  severity, 
and  an  extensive  area  a  severe  infection. 

Most  cases  of  pseudomehibranes  and  exudates,  entirely  confined 
to  portions  of  the  tonsils  in  adults,  are  not  due  to  the  diphtheria  bacilli, 
although  a  few  cases  presenting  these  symptoms  are.  The  more  com- 
plete the  involvement  of  the  tonsils  the  more  apt  the  case  is  to  be  due 
to  them.  Cases  presenting  the  appearances  found  in  scarlet  fever,  in 
which  a  thin,  grayish  membrane  lines  the  borders  of  the  uvula  and 
faucial  pillars,  are  rarely  diphtheritic.  As  a  rule,  pseudomembranous 
inflammations  complicating  scarlet  fever,  syphilis,  and  other  infectious 
diseases  are  due  to  the  activity  of  the  pathogenic  cocci  and  other  bac- 
teria, induced  by  the  inflamed  condition  of  the  mucous  membranes 
due  to  the  scarlatinal  or  other  poison.  But  from  time  to  time  such 
cases,  if  they  have  been  exposed  to  diphtheria,  may  be  complicated 
by  it,  and  in  some  epidemics  mixed  infection  is  common. 

Exudate  Due  to  the  Diphtheria  Bacilli  Contrasted  with  that  Due  to 
other  Bacteria. — As  a  rule,  the  exudate  in  diphtheria  is  firmly  incor- 
porated with  the  underlying  mucous  membrane,  and  cannot  be  removed 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     217 

without  leaving  a  bleeding  surface,  at  least  until  convalescence.  The 
tissues  surrounding  the  exudate  are  more  or  less  inflamed  and  swollen. 
Where  other  bacteria  produce  the  irritant  the  exudate,  except  in  cases 
due  to  the  bacillus  described  by  Vincent,  is  usually  loosely  attached, 
collected  in  small  masses,  and  easily  removable.  Exceptions,  how- 
ever, occur  in  both  these  diseases,  so  that  in  true  diphtheria  the  exudate 
may  be  easily  removed,  and  in  lesions  due  to  other  bacteria  the  exudate 
may  be  firmly  adherent. 

Paralysis  following  a  pseudomembranous  inflammation  is  an  almost 
positive  indication  that  the  case  was  one  of  diphtheria,  although  slight 
paralysis  has  followed  in  a  very  few  cases  in  which  careful  cultures 
revealed  no  diphtheria  bacilli.  These,  if  not  true  diphtheria,  must  be 
considered  very  exceptional  cases. 

Bacteriological  Diagnosis. — From  the  above  it  is  apparent  that  fully 
developed  characteristic  cases  of  diphtheria  are  readily  diagnosticated, 
but  that  many  of  the  less  marked,  or  at  an  early  period  undeveloped, 
cases  are  difficult  to  differentiate  the  one  from  the  other.  In  these 
cases  cultures  are  of  the  utmost  value,  since  they  enable  us  to  isolate 
those  in  which  the  bacilli  are  found,  and  to  give  preventive  infections 
of  antitoxin  to  both  the  sick  and  those  in  contact  with  them,  if  this  has 
not  already  been  done.  As  a  rule,  cultures  do  not  give  us  as  much 
information  as  to  the  gravity  of  the  case  as  the  clinical  appearances, 
for  by  the  end  of  twenty-four  to  forty-eight  hours  the  extent  of  the 
disease  is  usually  possible  of  determination.  The  reported  absence 
of  bacilli  in  a  culture  must  be  given  weight  in  proportion  to  the  skill 
with  which  the  culture  was  made,  the  suitableness  of  the  media,  the 
location  of  the  disease,  and  the  knowledge  and  experience  of  the  one 
who  examined  it. 

Diphtheria  does  not  occur  without  the  presence  of  the  diphtheria 
bacilli;  but  there  have  been  many  cases  of  diphtheria  in  which,  for  one 
or  another  reason,  no  bacilli  were  found  in  the  cultures  by  the  examiner. 
In  many  of  these  cases  later  cultures  revealed  them.  In  a  convalescent 
case  the  absence  of  bacilli  in  any  one  culture  indicates  that  there  are 
certainly  not  many  bacilli  left  in  the  throat.  Only  repeated  cultures 
can  prove  their  total  absence. 

TECHNIQUE  OF  THE  BACTERIOLOGICAL  DIAGNOSIS.  Collection  of 
the  Blood  Serum  and  its  Preparation  for  Use  in  Cultures. — A  covered 
glass  jar,  which  has  been  thoroughly  cleansed  with  hot  water,  is  taken 
to  the  slaughter-house  and  filled  with  freshly  shed  blood  from  a  calf  or 
sheep.  The  blood  is  received  directly  in  the  jar  as  it  spurts  from  the 
cut  in  the  throat  of  the  animal.  After  the  edge  of  the  jar  has  been 
wiped  it  is  covered  with  the  lid  and  set  aside,  where  it  may  stand  quietly 
until  the  blood  has  thoroughly  clotted.  The  jar  is  then  carried  to  the 
laboratory  and  placed  in  an  ice-chest.  If  the  jar  containing  the  blood 
is  carried  about  before  the  latter  has  clotted,  very  imperfect  separation 
of  the  serum  will  take  place.  It  is  well  to  inspect  the  blood  in  the  jar 
after  it  has  been  standing  a  few  hours,  and,  if  the  clot  is  found  adhering 
to  the  sides,  to  separate  it  by  a  rod.  The  blood  is  allowed  to  remain 


218  BACTERIA  PATHOGENIC  TO  MAN 

twenty-four  hours  on  the  ice,  and  then  the  serum  which  surrounds  the 
clot  is  syphoned  off  by  a  rubber  tube  and  mixed  with  one-third  its  quan- 
tity of  nutrient  beef-broth,  to  which  1  per  cent,  glucose  has  been  added. 
This  constitutes  the  Loeffler  blood-serum  mixture.  This  is  poured 
into  tubes,  which  should  be  about  four  inches  in  length  and  one-half 
of  an  inch  in  diameter,  having  been  previously  plugged  with  cotton 
and  sterilized  by  dry  heat  at  150°  C.  for  one  hour.  Care  should  be 
taken  in  filling  the  tubes  to  avoid  the  formation  of  air  bubbles,  as  they 
leave  a  permanently  uneven  surface  when  the  serum  has  been  coagu- 
lated by  heat.  To  prevent  this  the  end  of  the  pipette  or  funnel  which 
contains  the  serum  should  be  inserted  well  into  the  test-tube.  About 
3  c.c.  are  sufficient  for  each  tube  if  the  small  size  is  employed,  if 
not  5  c.c.  are  required.  The  tubes,  having  been  filled  to  the 
required  height,  are  now  to  be  coagulated  and  sterilized.  They 
are  placed  slanted  at  the  proper  angle  and  then  kept  for  two  hours 
at  a  temperature  just  below  95°  C.  For  this  purpose  a  Koch  serum 
coagulator  or  a  double  boiler  serves  best,  though  a  steam  sterilizer 
will  suffice.  If  the  latter  is  used  a  wire  frame  must  be  arranged  to  hold 
the  tubes  at  the  proper  inclination,  and  the  degree  of  heat  must  be 
carefully  watched,  as  otherwise  the  temperature  may  go  too  high,  and 
if  the  serum  is  actually  boiled  the  culture  medium  will  be  spoiled.  After 
sterilization  by  this  process  the  tubes  containing  the  sterile,  solidified 
blood  serum  can  be  placed  in  covered  tin  boxes,  or  stopped  with  sterile 
paraffined  corks  and  kept  for  months.  The  serum  thus  prepared  is 
quite  opaque  and  firm. 

Swab  for  Inoculating  Culture  Tubes. — The  swab  we  use  to  inocu- 
late the  serum  is  made  as  follows :  A  stiff,  thin,  iron  rod,  six  inches  in 
length,  is  roughened  at  one  end  by  a  few  blows  of  a  hammer,  and  about 
this  end  a  little  absorbent  cotton  is  firmly  wound.  Each  swab  is  then 
placed  in  a  separate  glass  tube,  and  the  mouths  of  the  tubes  are  plugged 
with  cotton.  The  tubes  and  rods  are  then  sterilized  by  dry  heat  at 
about  150°  C.  for  one  hour,  and  stored  for  future  use.  These  cotton 
swabs  have  proved  much  more  serviceable  for  making  inoculations 
than  platinum-wire  needles  or  wooden  sticks,  especially  in  young  chil- 
dren and  in  laryngeal  cases.  It  is  easier  to  use  the  cotton  swab  in  such 
cases,  and  it  gathers  up  so  much  more  material  for  the  inoculation 
that  it  has  seemed  more  reliable. 

For  convenience  and  safety  in  transportation  "culture  outfits"  have 
been  devised,  which  consist  usually  of  a  small  wooden  box  containing 
a  tube  of  blood  serum,  a  tube  holding  a  swab,  and  a  record  blank. 
These  "culture  outfits"  may  be  carried  or  sent  by  messenger  or  express 
to  any  place  desired. 

Directions  for  Inoculating  Culture  Tubes  with  the  Exudate. — The 
patient  is  placed  in  a  good  light,  and,  if  a  child,  properly  held.  The 
swab  is  removed  from  its  tube,  and,  while  the  tongue  is  depressed  with 
a  spoon,  is  passed  into  the  pharynx  (if  possible,  without  touching  the 
tongue  or  other  parts  of  the  mouth)  and  is  rubbed  gently  but  firmly 
against  any  visible  membrane  on  the  tonsils  or  in  the  pharynx,  and 


THE  BACILLUS  AXD  THE  BACTERIOLOGY  OF  DIPHTHERIA     219 

then,  without  being  laid  down,  the  swab  is  immediately  inserted  in 
the  blood-serum  tube,  and  the  portion  which  has  previously  been  in 
contact  with  the  exudate  is  rubbed  a  number  of  times  back  and  forth 
over  the  whole  surface  of  the  serum.  This  should  be  done  thoroughly, 
but  it  is  to  be  gently  done,  so  as  not  to  break  the  surface  of  the  serum. 
The  swab  should  then  be  placed  in  its  tube,  and  both  tubes,  thin  cotton 
plugs  having  been  inserted,  are  reserved  for  examination  or  sent  to 
the  laboratory  or  collecting  station  (as  in  New  York  City).  If  sent 
to  the  health  department  laboratories  for  examination  the  blank  forms 
of  report  which  usually  accompany  each  "  outfit"  should  be  filled  out 
and  forwarded  with  the  tubes. 

Where  there  is  no  visible  membrane  (it  may  be  present  in  the  nose 
or  larynx)  the  swab  should  be  thoroughly  rubbed  over  the  mucous 
membrane  of  the  pharynx  and  tonsils,  and  in  the  nasal  cavities,  and  a 
culture  made  from  these.  In  very  young  children  care  should  be  taken 
not  to  use  the  swab  when  the  throat  contains  food  or  vomited  matter, 
as  then  the  bacteriological  examination  is  rendered  more  difficult. 
Under  no  conditions  should  any  attempt  be  made  to  collect  the  material 
shortly  after  the  application  of  strong  disinfectants  (especially  solu- 
tions of  corrosive  sublimate)  to  the  throat. 

Examination  of  Cultures. — The  culture  tubes  which  have  been 
inoculated,  as  described  above,  are  kept  in  an  incubator  at  37°  C.  for 
twelve  hours,  and  are  then  ready  for  examination.  W7hen  great  haste 
is  required,  even  five  hours  will  often  suffice  for  a  sufficient  growth  of 
bacteria  for  a  skilled  examiner  to  decide  as  to  the  presence  or  absence 
of  the  bacilli.  On  inspection  it  will  be  seen  that  the  surface  of  the 
blood  serum  is  dotted  with  numerous  colonies,  which  are  just  visible. 
No  diagnosis  can  be  made  from  simple  inspection;  if,  however,  the 
serum  is  found  to  be  liquefied  or  shows  other  evidences  of  contamina- 
tion the  examination  will  probably  be  unsatisfactory. 

In  order  to  make  a  microscopic  preparation  a  clean  platinum 
needle  is  inserted  in  the  tube  and  quite  a  large  number  of  colonies  are 
swept  writh  it  from  the  surface  of  the  culture  medium,  a  part  being 
selected  where  small  colonies  only  are  found.  A  sufficient  amount  of 
the  bacteria  adherent  to  the  needle  is  washed  off  in  the  drop  of  water 
previously  placed  on  the  cover-glass  and  smeared  over  its  surface. 
The  bacteria  on  the  glass  are  then  allowed  to  dry  in  the  air.  The  cover- 
glass  is  then  passed  quickly  through  the  flame  of  a  Bunsen  burner  or 
alcohol  lamp,  three  times  in  the  usual  way,  covered  with  a  few  drops 
of  Loeffler's  solution  of  alkaline  methylene  blue,  and  left  w  ithout  heat- 
ing for  five  to  ten  minutes.  It  is  then  rinsed  off  in  clear  water,  dried, 
and  mounted  in  balsam.  When  other  methods  of  staining  are  desired 
they  are  carried  out  in  the  proper  way. 

In  the  great  majority  of  cases  one  of  two  pictures  will  be  seen  with 
the  TV  oil-immersion  lens — either  an  enormous  number  of  character- 
istic Loeffler  bacilli,  with  a  moderate  number  of  cocci,  or  a  pure  culture 
of  cocci,  mostly  in  pairs  or  short  chains.  (See  Streptococcus.)  In  a  few 
cases  there  will  be  an  approximately  even  mixture  of  Loeffler  bacilli 


220  BACTERIA  PATHOGENIC  TO  MAN 

and  cocci,  and  in  others  a  great  excess  of  cocci.  Besides  these,  there 
will  be  occasionally  met  preparations  in  which,  with  the  cocci,  there 
are  mingled  bacilli  more  or  less  resembling  the  Loeffler  bacilli.  These 
bacilli,  which  are  usually  of  the  pseudodiphtheria  type  of  bacilli  (see 
Fig.  63),  are  especially  frequent  in  cultures  from  the  nose. 

In  not  more  than  one  case  in  twenty  will  there  be  any  serious  difficulty 
in  making  the  diagnosis,  if  the  serum  in  the  tube  was  moist  and  had 
been  properly  inoculated.  In  such  a  case  another  culture  must  be 
made  or  the  bacilli  plated  out  and  tested  in  pure  culture. 

Direct  Microscopic  Examination  of  the  Exudate. — An  immediate 
diagnosis  without  the  use  of  cultures  is  often  possible  from  a  micro- 
scopic examination  of  the  exudate.  This  is  made  by  smearing  a 
slide  or  cover-glass  with  a  little  of  the  exudate  from  the  swab,  drying, 
heating,  staining,  and  examining  it  microscopically.  This  examination, 
however,  is  much  more  difficult,  and  the  results  are  more  uncertain 
than  when  the  covers  are  prepared  from  cultures.  The  bacilli  from 
the  membrane  are  usually  less  typical  in  appearance  than  those  found 
in  cultures,  and  they  are  mixed  with  fibrin,  pus,  and  epithelial  cells. 
They  may  also  be  very  few  in  number  in  the  parts  reached  by  the  swab, 
or  bacilli  may  be  met  with  which  closely  resemble  the  Loeffler  bacilli  in 
appearance,  but  which  differ  greatly  in  growth  and  in  other  charac- 
teristics, and  have  absolutely  no  connection  with  them.  When  in  a 
smear  containing  mostly  cocci  a  few  of  these  doubtful  bacilli  are  present, 
it  is  impossible  either  to  exclude  or  to  make  the  diagnosis  of  diphtheria 
with  certainty.  Although  in  some  cases  this  immediate  examination 
may  be  of  the  greatest  value,  it  is  not  a  method  suitable  for  general 
use,  and  should  always  be  controlled  by  cultures. 

When  carried  out  in  the  best  manner  an  experienced  bacteriologist 
may  obtain  remarkably  accurate  results.  Higley  in  New  York  in  a 
series  of  consecutive  throat  cases  made  the  same  diagnosis  from  the 
direct  examination  of  smears  as  the  Health  Department  laboratory 
made  from  the  culture.  To  get  the  exudate  he  used  a  probe  armed 
with  a  loop  of  heavy  copper  wire  which  has  been  so  flattened  as  to 
act  as  a  blunt  curette.  He  makes  thus  thin  smears  from  the  exudate. 
After  drying  and  fixing  by  heat  the  smears  are  stained  for  five  seconds 
in  a  solution  made  by  adding  five  drops  of  Kiihnes  carbolic  methylene 
blue  in  7  c.c.  of  tap-water.  After  washing  and  drying  stain  for  one 
minute  in  a  solution  of  10  drops  of  carbol-fuchsin  in  7  c.c.  of 
water.  The  dilute  solution  should  be  freely  prepared.  The  diphtheria 
bacilli  will  appear  as  dark-red  or  violet  rods,  and  their  contour, 
mode  of  division,  and  arrangement  are  manifest. 

Animal  Inoculation  as  a  Test  of  Virulence. — If  the  determination  of 
the  virulence  of  the  bacilli  found  is  of  importance,  animal  inoculations 
must  be  made.  Experiments  on  animals  form  the  only  method  of 
determining  with  certainty  the  virulence  of  the  diphtheria  bacillus. 
For  this  purpose,  alkaline  broth  cultures  of  forty-eight  hours'  growth 
should  be  used  for  the  subcutaneous  inoculation  of  guinea-pigs.  The 
amount  injected  should  not  be  more  than  one-fifth  per  cent,  of  the  body- 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  DIPHTHERIA     221 


of  the  animal  inoculated,  unless  controls  with  antitoxin  are  made. 
In  the  large  majority  of  cases,  when  the  bacilli  are  virulent,  this  amount 
causes  death  within  seventy-two  hours.  At  the  autopsy  the  character- 
istic lesions  already  described  are  found.  Bacilli  which  in  cultures 
and  in  animal  experiments  have  shown  themselves  to  be  characteristic 
may  be  regarded  for  practical  purposes  as  certainly  true  diphtheria 
bacilli,  and  as  capable  of  producing  diphtheria  in  man  under  favorable 
conditions. 

For  an  absolute  test  of  specific  virulence  antitoxin  must  be  used. 
A  guinea-pig  is  injected  with  antitoxin,  and  then  this  and  a  control 
animal,  with  2  c.c.  of  a  broth  culture  of  the  bacilli  to  be  tested, 
if  the  guinea-pig  which  received  the  antitoxin  lives,  while  the  control 
dies,  it  was  surely  a  diphtheria  bacillus  which  killed  by  means  of  diph- 
theria toxin  —  or,  in  other  words,  not  simply  a  virulent  bacillus,  but  a 
virulent  diphtheria  bacillus.  When  the  bacilli  to  be  tested  grow  poorly 
in  a  simple  nutrient  bouillon  they  should  be  grown  in  bouillon  to  which 
one-third  its  quantity  of  ascitic  fluid  has  been  added.  Quite  a  number 
of  bacilli  have  been  met  with  which  killed  250-grm.  guinea-pigs  in 
doses  of  2  to  15  c.c.,  and  yet  were  unaffected  by  antitoxin.  These 
bacilli,  though  slightly  virulent  to  guinea-pigs,  produce  no  diphtheria 
toxin,  and  so  cannot,  to  the  best  of  our  belief,  produce  diphtheria  in 
man. 


CHAPTER  XVIII. 

THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  TETANUS. 

TETANUS  is  a  disease  which  is  characterized  by  a  gradual  onset  of 
general  spasm  of  the  voluntary  muscles,  commencing  in  man  most 
often  in  those  of  the  jaw  and  neck,  and  extending  in  severe  cases  to  all 
the  muscles  of  the  body.  The  disease  is  usually  associated  with  a 
wound  received  from  four  to  fourteen  days  previously. 

In  1884  Nicolaier,  under  Fliigge's  direction,  produced  tetanus  in 
mice  and  rabbits  by  the  subcutaneous  inoculation  of  particles  of  garden 
earth.  The  Italians,  Carle  and  Rattone,  had  just  before  demonstrated 
that  the  pus  of  an  infected  wound  from  a  person  attacked  with  tetanus 
could  produce  the  same  disease  in  rabbits,  and  showed  that  the  disease 
was  transmissible  by  inoculation  from  these  animals  to  others.  Finally, 
Kitasato,  in  1889,  obtained  the  bacillus  of  tetanus  in  pure  culture  and 
described  his  method  of  obtaining  it  and  its  biological  characters. 

Occurrence  in  Soil,  etc. — The  tetanus  bacillus  occurs  in  nature  as  a 
common  inhabitant  of  the  soil,  at  least  in  places  where  manure  has 
been  thrown,  being  abundant  in  many  localities,  not  only  in  the  super- 
ficial layers,  but  also  at  the  depth  of  several  feet.  It  has  been  found  in 
many  different  substances  and  places — in  hay-dust,  in  horse  and  cow 
manure,  in  the  mortar  of  old  masonry,  in  the  dust  from  horses'  hair; 
in  the  dust  in  rooms  of  houses,  barracks,  and  hospitals;  in  the  air,  and 
in  the  arrow  poison  of  certain  savages  in  the  New  Hebrides,  who  obtained 
it  by  smearing  the  arrow-heads  with  dirt  from  crab  holes  in  the  swamps. 

Morphology. — In  young  gelatin  cultures  the  bacilli  appear  as  motile, 
slender  rods,  with  rounded  ends,  0.5/*  to  0.8/t  in  diameter  by  2tu  to  4// 
in  length,  usually  occurring  singly,  but,  especially  in  old  cultures,  often 
growing  in  long  threads.  They  form  round  spores,  thicker  than  the 
cell  (from  I/*  to  1.5/J.  in  diameter),  occupying  one  of  its  extremities  and 
giving  to  the  rods  the  appearance  of  small  pins  (Fig.  84). 

Staining. — It  is  stained  with  the  ordinary  aniline  dyes,  and  is  not 
decolorized  by  Gram's  method.  The  spores  are  readily  stained  and 
may  be  demonstrated  by  double-staining  with  Ziehl's  method.  The 
flagella  are  fairly  easily  stained  in  very  young  cultures. 

Biology. — An  anaerobic,  liquefying,  moderately  motile  bacillus.  It 
has  abundant  flagella.  Forms  spores,  and  in  the  spore  stage  it  is  not 
motile.  It  grows  slowly  at  temperatures  from  20°  to  24°  C.,  and  best 
at  37°  C.,  when  within  twenty-four  hours  it  forms  spores.  It  will  not 
in  pure  culture  grow  in  the  presence  of  oxygen  or  carbon  dioxide  gas, 
but  grows  well  in  an  atmosphere  of  hydrogen  gas.  With  certain  other 
bacteria  the  tetanus  bacillus  grows  luxuriantly  in  the  presence  of  oxygen. 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  TETANUS      223 

Growth  in  Media. — The  bacillus  of  tetanus  grows  in  ordinary  nutrient 
gelatin  and  agar  of  a  slightly  alkaline  reaction.  The  addition  to  the 
media  of  1.5  per  cent,  of  glucose  causes  the  development  to  be  more 
rapid  and  abundant.  It  also  grows  abundantly  in  alkaline  bouillon 
under  an  atmosphere  of  hydrogen.  On  gelatin  plates  the  colonies 
develop  slowly;  they  resemble  somewhat  the  colonies  of  the  bacillus 
xitlitiliff,  and  have  a  dense,  opaque  centre  surrounded  by  fine,  diverg- 
ing rays.  Liquefaction  takes  place  more  slowly,  however,  than  with 
the  bacillus  subtilis,  and  the  resemblance  to  these  colonies  is  soon 
lost. 

The  colonies  on  agar  are  quite  characteristic.  To  the  naked  eye 
they  present  the  appearance  of  light,  fleecy  clouds;  under  the  micro- 
scope, a  tangle  of  fine  threads. 

The  stab  cultures  in  gelatin  exhibit  the  appearance  of  a  cloudy,  linear 
mass,  with  prolongations  radiating  into  the  gelatin  from  all  sides. 
Liquefaction  takes  place  slowly,  generally  with  the  production  of  gas. 
In  stab  cultures  in  agar  a  growth  occurs  not  unlike  in  structure  that  of 

FIG.  84 


4 


£•' 


Tetanus  bacilli  with  spores  in  distended  ends.    X  1100  diameters. 

a  miniature  pine-tree.  Alkaline  bouillon  is  rendered  somewhat  turbid 
by  the  growth  of  the  tetanus  bacillus.  In  all  cases  a  production  of  gas 
results,  accompanied  by  a  characteristic  and  very  disagreeable  odor. 
It  develops  in  milk  without  coagulating  it. 

Resistance  of  Spores  to  Deleterious  Influences. — The  spores  of  the 
tetanus  bacillus  are  very  resistant  to  outside  influences;  in  a  desic- 
cated condition  they  retain  their  vitality  for  years,  and  are  not  destroyed 
in  two  and  a  half  months  when  present  in  putrefying  material.  They 
withstand  an  exposure  of  one  hour  to  80°  C.,  but  are  killed  by  an  ex- 
posure of  five  minutes  at  100°  C.  to  live  steam.  They  resist  the  action 
of  5  per  cent,  carbolic  acid  for  ten  hours.  A  5  per  cent,  solution  of 
carbolic  acid,  however,  to  which  0.5  per  cent,  of  hydrochloric  acid  has 
been  added,  destroys  them  in  two  hours.  They  are  killed  when  acted 
upon  for  three  hours  by  bichloride  of  mercury  (1: 1000),  and  in  thirty 
minutes  when  0.5  per  cent.  HC1  is  added  to  the  solution.  Silver  nitrate 
solutions  destroy  the  spores  in  one  minute  in  1  per  cent,  solution  and 
in  about  five  minutes  in  1 : 1000  solution. 


224  BACTERIA  PATHOGENIC  TO  MAN 

Isolation  of  Pure  Cultures. — The  growth  of  the  tetanus  bacillus  in  the 
animal  body  is  comparatively  scanty,  and  is  usually  associated  with 
that  of  other  bacteria;  hence,  the  organism  is  difficult  to  obtain  in  pure 
culture.  The  method  of  procedure  proposed  by  Kitasato,  which,  how- 
ever, is  not  always  successful,  consists  in  inoculating  slightly  alkaline 
nutrient  agar  or  glucose  bouillon  with  the  tetanus-bearing  material 
(pus  or  tissue  from  the  inoculation  wound),  keeping  the  culture  under 
anaerobic  conditions  for  twenty-four  to  forty-eight  hours  at  a  tempera- 
ture of  37°  C.,  and,  after  the  tetanus  spores  have  formed,  heating  it 
for  one-half  an  hour  at  80°  C.,  to  destroy  the  associated  bacteria. 
The  spores  of  the  tetanus  bacillus  are  able  to  survive  this  exposure, 
so  that  when  anaerobic  cultures  are  then  made  in  the  usual  way  the 
tetanus  colonies  develop.  When  the  tetanus  bacilli  are  the  only  spore- 
bearing  bacteria  present,  pure  cultures  are  readily  obtained ;  when  other 
spore-bearing  anaerobic  bacteria  are  present,  the  isolation  of  a  pure 
culture  may  be  a  matter  of  difficulty. 

Pathogenesis. — In  mice,  guinea-pigs,  rabbits,  horses,  goats,  and  a 
number  of  other  animals  inoculations  of  pure  cultures  of  the  tetanus 
bacillus  cause  typical  tetanus  after  an  incubation  of  from  one  to  three 
days.  A  mere  trace  of  an  old  culture — only  as  much  as  remains  cling- 
ing to  a  platinum  needle — is  often  sufficient  to  kill  very  susceptible 
animals  like  mice  and  guinea-pigs.  Other  animals  require  a  larger 
amount.  Rats  and  birds  are  but  little  susceptible,  and  fowls  scarcely 
at  all.  Man  is  more  susceptible  than  any  of  the  animals  so  far  tested. 
A  horse  is  about  six  times  as  sensitive  as  a  guinea-pig  and  three 
hundred  thousand  times  as  sensitive  as  a  hen.  It  is  a  remarkable  fact 
that  an  amount  of  toxin  sufficient  to  kill  a  hen  would  suffice  to  kill  500 
horses.  It  is  estimated  that  if  1  gram  of  horse  requires  1  part  of  toxin 
to  kill,  then  1  gram  of  guiriea-pig  requires  6  parts,  1  of  mouse  12,  of 
goat  24,  of  dog  500,  of  rabbit  1500,  of  cat  6000,  of  hen  360,000. 
Cultures  from  different  cases  vary  greatly  in  their  toxicity.  On  the 
inoculation  of  less  than  a  fatal  dose  in  test  animals  a  local  tetanus 
may  be  produced,  which  lasts  for  days  and  wrecks  and  then  ends  in 
recovery.  On  killing  the  animal  there  is  found  at  autopsy,  just  at  the 
point  of  inoculation,  a  hemorrhagic  spot,  and  no  changes  other  than 
these  here  or  in  the  interior  organs.  A  few  tetanus  bacilli  may  be  detected 
locally  with  great  difficulty,  often  none  at  all;  possibly  a  few  may  be 
found  in  the  region  of  the  lymphatic  glands.  From  this  scanty  occur- 
rence of  bacilli  the  conclusion  has  been  reached  that  the  bacilli  of 
tetanus,  when  inoculated  in  pure  culture,  do  not  multiply  to  any  great 
extent  in  the  living  body,  but  only  produce  lesions  through  the  absorp- 
tion of  the  poison  which  they  develop  at  the  point  of  infection.  It  has 
been  found  that  pure  cultures  of  tetanus,  after  the  germs  have  sporulated 
and  the  toxins  been  destroyed  by  heat,  can  be  injected  into  animals 
without  producing  tetanus.  But  if  a  culture  of  non-pathogenic  organ- 
isms is  injected  simultaneously  with  the  spores,  or  if  there  is  an  effusion 
of  blood  at  the  point  of  injection,  or  if  there  was  a  previous  bruising 
of  the  tissues,  the  animals  surely  die  of  tetanus.  Even  irritating  foreign 


///A  BACILLUS  AND  THE  BACTERIOLOGY  OF  TETANUS      225 

bodies  have  been  introduced  along  with  the  spores  deprived  of  their 
toxins,  and  tetanus  did  not  develop;  but  if  the  wounds  containing  the 
foreign  bodies  became  infected  with  other  bacteria,  tetanus  developed 
and  the  animal  died.  From  such  experiments  it  seems  that  a  mixed 
infection  aids  greatly  in  the  development  of  tetanus  when  the  infection 
is  produced  by  spores. 

Natural  Infection. — Here  the  infection  may  be  considered  as  probably 
produced  by  the  bacilli  in  their  spore  state,  and  the  conditions  favoring 
infection  are  almost  always  present.  A  wound  of  some  kind  has 
occurred,  penetrating  at  least  through  the  skin,  though  perhaps  of  a 
most  trivial  character,  such  as  might  be  caused  by  a  dirty  splinter  of 
wood,  and  the  bacilli  or  their  spores  are  thus  introduced  from  the  soil 
in  which  they  are  so  widely  distributed.  If  in  any  given  case,  the  tissues 
being  healthy,  the  ordinary  saprophytic  germs  are  killed  by  proper  dis- 
infection at  once,  a  mixed  infection  does  not  take  place,  and  tetanus  will 
not  develop.  If,  however,  the  tissues  infected  be  badly  bruised  or 
lacerated,  the  spores  may  develop  and  produce  the  disease.  Gelatin  is 
found  to  contain  occasionally  tetanus  spores. 

Occurrence  and  Duration  of  Life  of  Tetanus  Bacilli. — With  regard  to  the 
persistence  of  tetanus  spores  upon  objects  where  they  have  found  a 
resting  place,  Henrijean  reports  that  by  means  of  a  splinter  of  wood 
which  had  once  caused  tetanus  he  was  able  after  eleven  years  to  again 
cause  the  disease  by  inoculating  an  animal  with  the  infective  material. 
The  bacilli  of  tetanus  are  apparently  more  numerous  in  certain  localities 
than  in  others — for  example,  some  parts  of  Long  Island  and  New  Jersey, 
have  become  notorious  for  the  number  of  cases  of  tetanus  caused  by 
small  wounds — but  they  are  very  generally  distributed,  as  the  experi- 
ments on  animals  inoculated  with  garden  earth  have  shown,  and  are 
fairly  common  in  New  York  City.  In  some  islands  and  countries  in 
the  tropics  puerperal  tetanus  and  tetanus  in  the  newborn  is  very  fre- 
quent. Tetanus  bacilli  are  found  in  intestines  of  about  10  per  cent,  of 
horses  and  calves  living  in  the  vicinity  of  New  York  City. 

Tetanus  in  Man. — Man  and  almost  all  domestic  animals  are  subject  to 
tetanus.  On  examination  of  an  infected  individual  very  little  local  evi- 
dence of  the  disease  can  be  discovered.  Generally  at  the  point  of 
infection,  if  there  is  an  external  wound,  some  pus  is  to  be  seen,  in 
which,  along  with  numerous  other  bacteria,  tetanus  bacilli  or  their  spores 
may  be  found.  Although  rather  deep  wounds  are  usually  the  seat  of 
infection,  at  times  such  superficial  wounds,  as  an  acne  pustule  or  a 
vaccination,  may  give  the  occasion  for  infection.  Not  only  undoubted 
traumatic  tetanus,  but  also  all  the  other  forms  of  tetanus,  are  now 
conceded  to  be  produced  by  the  tetanus  bacillus — puerperal  tetanus, 
tetanus  neonatorum,  and  idiopathic  tetanus.  In  tetanus  neonatonun 
infection  is  introduced  through  the  navel,  in  puerperal  tetanus  through 
the  inner  surface  of  the  uterus.  It  should  be  borne  in  mind  that  when 
there  is  no  external  and  visible  wound  there  may  be  an  internal  one. 

Toxins  of  the  Tetanus  Bacillus. — It  is  evident  from  the  localization 
of  the  tetanus  bacilli  at  the  point  of  inoculation  and  their  slight  mul- 

15 


226  BACTERIA  PATHOGENIC  TO  MAN 

tiplication  at  this  point  that  they  exert  their  action  through  the  produc- 
tion of  powerful  toxins.  These  toxins  are  named,  according  to  their 
action,  the  tetanospasmin  and  the  tetanolysin.  One  one-hundredth  of 
a  milligram  of  the  filtrate  of  an  eight-day  glucose  bouillon  culture  of 
a  fully  virulent  bacillus  is  sufficient  to  kill  a  mouse.  From  this  filtrate, 
however,  the  active  toxic  substance  has  been  obtained  in  a  much  more 
concentrated  form.  The  purified  and  dried  tetanus  toxin  prepared  by 
Brieger  and  Cohn  was  surely  fatal  to  a  15-gram  mouse  in  a  dose  of 
0.00000005  gram.  Reckoning  according  to  the  body- weight  of  75  kilo- 
grams, or  175  pounds,  it  would  require  but  0.00023  gram,  or  0.23 
milligram,  of  this  toxin  to  prove  fatal  to  a  man.  The  appalling  strength 
of  tetanus  toxin  may  readily  be  appreciated.  By  comparing  it  with 
snake  poison  and  strychnine,  Calmette  has  found  that  dried  cobra  venom 
would  require  4.375  milligrams  to  kill  a  man  of  70  kilograms.  A  fatal 
dose  of  strychnine  is  from  30  to  100  milligrams. 

The  quantity  of  the  toxin  produced  in  nutrient  media  varies  accord- 
ing to  the  age  of  the  culture,  the  composition  of  the  culture  fluid,  reac- 
tion, completeness  of  the  exclusion  of  oxygen,  etc.  The  variation  in 
strength  is  partly  due  to  the  extreme  sensitiveness  of  the  toxin,  which 
deteriorates  on  keeping  or  on  exposure  to  light,  being  also  sensibly 
affected  by  most  chemical  reagents  and  destroyed  by  heating  to  55°  to 
60°  C.  for  any  length  of  time.  It  retains  its  strength  best  when  protected 
from  heat,  light,  oxygen,  and  moisture.  Under  the  best  conditions  the 
amount  of  toxin  produced  in  cultures  by  the  fifth  day  is  such  that 
0.000005  c.c.  is  the  fatal  dose  for  a  15-gram  mouse. 

The  tetanus  cultures  retain  their  ability  to  produce  toxins  unaltered 
when  kept  under  suitable  conditions ;  but  when  subjected  to  deleterious 
influences  they  may  entirely  lose  it.  The  usual  medium  for  the  develop- 
ment of  the  toxin  is  a  slightly  alkaline  bouillon  containing  1  per  cent,  of 
peptone  and  0.5  per  cent.  salt.  The  addition  of  more  than  a  trace  of 
sugar  or  glycerin  is  to  be  avoided,  as  the  acid  produced  injures  the  toxin. 

Action  of  Tetanus  Toxin  in  the  Body. — After  the  absorption  of  the 
poison  there  is  a  lapse  of  time  before  any  effects  are  noticed.  With 
an  enormous  amount,  such  as  30,000  fatal  doses,  this  is  about  twelve 
hours;  with  ten  fatal  doses,  thirty-six  to  forty-eight  hours;  with  two  fatal 
doses,  two  to  three  days.  Less  than  a  fatal  dose  will  produce  local 
symptoms.  The  parts  first  to  be  affected  with  tetanus  are,  in  about 
one-third  of  the  cases  in  man  and  usually  in  animals,  the  muscles 
lying  in  the  vicinity  of  the  inoculation — for  instance,  the  hind  foot  of 
a  mouse  inoculated  on  that  leg  is  first  affected,  then  the  tail,  the  other 
foot,  the  back  and  chest  muscles  on  both  sides,  and  the  forelegs,  until 
finally  there  is  a  general  tetanus  of  the  entire  body.  In  mild  cases,  or 
when  a  dose  too  small  to  be  fatal  has  been  received,  the  tetanic  spasm 
may  remain  confined  to  the  muscles  adjacent  to  the  point  of  inoculation 
or  infection.  The  symptoms  following  a  fatal  dose  of  toxin  vary  greatly 
with  the  method  of  injection.  Intraperitoneal  injection  is  followed  by 
symptoms  which  can  hardly  be  distinguished  from  those  due  to  many 
other  poisons.  Injection  into  the  brain  is  followed  by  restlessness  and 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  TETANUS     227 

epileptiform  convulsions.  The  tetanus  toxins  undoubtedly  combine 
readily  with  the  cells  of  the  central  nervous  system.  They  also  com- 
bine with  other  tissue  cells  with  less  apparent  effects.  The  symptoms 
in  tetanus  depend  upon  an  increased  reflex  excitability  of  the  motor 
cells  of  the  spinal  cord,  the  medulla,  and  pons. 

Presence  of  Tetanus  Toxin  in  the  Blood  of  Infected  Animals. — The  blood 
usually  contains  the  poison,  as  has  been  proved  experimentally  on  animal. 
Neisser  showed  that  the  blood  of  a  tetanic  patient  was  capable  of  inducing 
tetanus  in  animals  when  injected  subcutaneously.  In  St.  Louis  the  serum 
of  a  horse  dying  of  tetanus  was  given  by  accident  in  doses  of  5  to  10  c.c.  to 
a  number  of  children,  with  the  development  of  fatal  tetanus.  In  this 
connection  Bolton  and  Fisch  showed  by  a  series  of  experiments  that 
much  toxin  might  accumulate  in  the  serum  before  symptoms  became 
marked.  Kitasato  also  found  the  serous  exudates  of  the  pleural  and 
pericardial  cavities  as  well  as  the  blood  of  tetanic  animals  would  cause 
tetanus  when  transferred  to  other  animals.  Ehrlich  has  shown  that 
besides  the  predominant  poison  which  gives  rise  to  spasm  (tetano- 
spasmin)  there  exists  a  poison  capable  of  producing  solution  of  certain 
red  blood  corpuscles.  This  he  calls  tetanolysin.  It  was  not  found  in 
all  culture  fluids  and  does  not  pass  through  in  the  first  portion  of  the 
filtrate  from  a  porcelain  filter.  Whether  in  actual  disease  this  poison  is 
ever  in  sufficient  amount  to  cause  appreciable  harm  is  not  known. 

Tetanus  Antitoxin. — Behring  and  Kitasato  were  the  first  to  show 
the  possibility  of  immunizing  animals  against  tetanus  infection.  The 
entire  procedure  is  analogous  to  immunization  against  diphtheria.  The 
treatment  of  tetanus  is  directed  against  the  action  of  the  toxin  and  this 
is  accomplished  by  the  neutralization  of  the  toxin  by  antitoxin  in  the 
body. 

The  immunizing  experiments  in  tetanus  have  borne  practical  fruit, 
for  it  was  through  them  that  the  principle  of  serum  therapeutics  first 
became  known — the  protective  and  curative  effects  of  the  blood  serum 
of  immunized  animals.  It  was  found  that  animals  could  be  pro- 
tected from  tetanus  infection  by  the  previous  or  simultaneous  injection 
of  tetanus  antitoxin,  provided  that  such  antitoxic  serum  was  obtained 
from  a  thoroughly  immunized  animal.  From  this  it  was  assumed 
that  the  same  result  could  be  produced  in  natural  tetanus  in  man. 
Unfortunately,  however,  the  conditions  in  the  natural  disease  are  very 
much  less  favorable,  inasmuch  as  treatment  is  usually  commenced  not 
shortly  after  the  infection  has  taken  place,  but  only  on  the  appearance 
of  tetanic  symptoms,  when  the  poison  has  already  attacked  the  cells  of 
the  central  nervous  system. 

The  tetanus  antitoxin  is  developed  in  the  same  manner  as  the  diph- 
theria antitoxin — by  inoculating  the  tetanus  toxin  in  increasing  doses 
into  horses.  The  toxin  is  produced  in  bouillon  cultures  grown  anae'ro- 
birally.  After  six  to  fifteen  days  the  culture  fluid  is  filtered  through 
porcelain,  and  the  germ-free  filtrate  is  used  for  the  inoculations.  The 
horses  receive  \  c.c.  as  the  initial  dose  of  a  toxin  of  which  1  c.c.  kills 
250,000  grams  of  guinea-pig,  and  along  with  this  a  sufficient  amount 


228  BACTERIA  PATHOGENIC  TO  MAN 

of  antitoxin  to  neutralize  it.  In  five  days  this  dose  is  doubled,  and 
then  every  five  to  seven  days  larger  amounts  are  given.  After  the 
third  injection  the  antitoxin  is  omitted.  The  dose  is  increased  as 
rapidly  as  the  horses  can  stand  it,  until  they  support  700  to  800  c.c.  or 
more  at  a  time.  This  amount  should  not  be  injected  in  a  single  place, 
or  severe  local  and  perhaps  fatal  local  tetanus  may  develop.  After  some 
months  of  this  treatment  the  blood  of  the  horse  contains  the  antitoxin 
in  sufficient  amount  for  therapeutic  use.  When  the  animals'  tempera- 
tures are  normal  and  they  have  recovered  from  the  dose  of  toxin  last 
given,  they  are  bled  into  sterile  flasks  and  the  serum  collected.  A  quicker 
and  safer  method  of  immunization  is  to  mix  antitoxin  with  larger  doses 
of  toxin  in  the  first  four  injections. 

Technique  of  Testing  Antitoxin  Serum  for  Value  in  Antitoxin. — Tetanus 
antitoxin  is  tested  exactly  in  the  same  manner  as  diphtheria  antitoxin,, 
except  that  the  unit  adopted  by  different  dispensers  is  different.  This 
confuses  the  physician  and  almost  compels  him  to  give  a  certain  number 
of  cubic  centimetres  without  regard  to  the  units  contained.  The  amount 
of  antitoxic  serum  which  neutralizes  an  amount  of  test  toxin  which 
would  destroy  40,000,000  grams  of  mouse  contains  1  unit  of  antitoxin 
by  the  German  standard.  One  large  American  producer  considers 
1  unit  of  antitoxin  to  be  the  amount  which  neutralizes  only  100  grams 
of  mouse.  In  the  French  method  the  amount  of  antitoxin  which  is 
required  to  protect  a  mouse  from  a  dose  of  toxin  sufficient  to  kill  in 
four  days  is  determined,  and  the  strength  of  the  antitoxin  is  stated 
by  determining  the  amount  of  serum  required  to  protect  1  gram  of 
animal.  If  0.001  c.c.  protected  a  10-gram  mouse  the  strength  of  that 
serum  would  be  1 : 10,000.  Guinea-pigs  are  sometimes  used  in  place 
of  mice.  The  United  States  government  should  make  an  official  tetanus 
antitoxin  unit  in  the  same  way  it  has  an  official  diphtheria  antitoxin 
unit.  The  toxin  used  for  testing  is  preserved  by  precipitating  it  with 
saturated  ammonium  sulphate  and  drying  and  preserving  the  pre- 
cipitate in  sealed  tubes.  As  required,  it  is  dissolved  in  10  per  cent, 
salt  solution  as  above  stated.  For  small  testing  stations  the  best  way  is 
to  obtain  some  freshly  standardized  antitoxin  and  compare  serums 
with  this. 

Persistence  of  Antitoxin  in  the  Blood. — Ransom  has  recently  shown 
that  the  tetanus  antitoxin,  whether  directly  injected  or  whether  produced 
in  the  body,  is  eliminated  equally  rapidly  from  the  blood  of  an  animal 
provided  that  the  serum  was  from  an  animal  of  the  same  species.  If 
from  a  different  species  it  is  much  more  quickly  eliminated.  From 
this  we  see  a  possible  explanation  of  the  fact  that  immunity  in  man, 
due  to  an  injection  of  the  antitoxic  serum  of  the  horse,  is  less  per- 
sistent than  immunity  conferred  by  an  attack  of  the  disease. 

The  same  author  found  some  interesting  facts  in  testing  the  antitoxic 
values  of  the  serum  of  an  immunized  mare,  of  its  foal,  and  of  the  milk. 
The  foal's  serum  was  one-third  the  strength  of  the  mare's,  and  one 
hundred  and  fifty  times  that  of  the  mare's  milk.  In  two  months  the 
mare's  serum  lost  two-thirds  in  antitoxic  strength,  the  foal's  five-sixths, 


///A  BACILLUS  AND  THE  BACTERIOLOGY  OF  TET.\.\rs      229 

and  the  milk  one-half.  Injections  of  toxin  were  then  given  the  mare,  so 
that  it  doubled  its  original  strength  in  one  month.  The  milk  increased 
eightfold,  but  the  foal's  continued  to  lose  in  antitoxin,  although  it  w;i> 
feeding  on  the  antitoxic  milk. 

Toxin  and  Antitoxin  in  the  Living  Organism.  Animal  Experiments. — 
Very  recently  the  studies  of  H.  Meyer  and  Ransom  as  well  as  those 
of  Marie  and  Morax  in  Roux's  laboratory  have  thrown  considerable 
light  on  the  much  disputed  phenomena  observed  in  poisoning  by  tetanus 
toxin.  The  investigations  of  Gumprecht  in  1895  had  made  it  highly 
probable  that  all  the  pathological  symptoms  were  due  to  a  poisoning 
of  the  central  nervous  system.  The  paths,  however,  by  which  the 
poison  reached  its  central  points  of  attack  were  still  doubtful.  The 
experiments  of  Meyer  and  Ransom  and  of  Marie  and  Morax  have 
practically  proved  that  the  poison  is  transported  to  the  central  nervous 
system  by  way  of  the  motor  nerves — and  by  no  other  channel.  So 
far  as  the  experiments  are  concerned,  I  must  refer  the  reader  to  the 
original.  These  show  that  the  essential  element  for  the  absorption 
and  transportation  of  the  toxin  is  not  the  nerve  sheath  or  the  lymph 
channels,  but  the  axis-cylinder,  the  intramuscular  endings  of  which 
the  toxin  penetrates.  The  poison  is  taken  up  quite  rapidly.  Marie 
and  Morax  were  able  to  demonstrate  the  poison  in  the  corresponding 
nerve  trunk  (sciatic)  one  and  a  half  hours  after  the  injection.  Absorp- 
tion, however,  and  conduction  are  dependent  to  a  large  extent  on  the 
nerves  being  intact.  A  nerve  cut  across  takes  very  much  longer  to 
take  up  the  poison  (about  twenty-four  hours),  and  a  degenerated  nerve 
takes  up  no  poison  whatever.  In  other  words,  we  see  that  section  of 
the  nerve  prevents  the  absorption  of  the  poison  by  way  of  the  nerve 
channels.  Similarly  section  of  the  spinal  cord  prevents  the  poison  from 
ascending  to  the  brain. 

According  to  Meyer  and  Ransom  the  reason  why  the  sensory  nerves 
do  not  play  any  role  in  the  conduction  of  the  poison  lies  in  the  presence 
of  the  spinal  ganglion,  which  places  a  bar  to  the  advance  of  the  poison. 
Injections  of  toxin  into  the  posterior  root  leads  to  a  tetanus  dolorosus, 
which  is  characterized  by  strictly  localized  sensitiveness  to  pain. 

Ascending  centripetally  along  the  motor  paths  the  poison  reaches 
the  motor  spinal  ganglia  on  the  side  of  inoculation;  then  it  affects  the 
ganglia  of  the  opposite  side,  making  them  hypersensitive.  The  visible 
result  of  this  is  the  highly  increased  muscle  tonus — i.  e.,  rigidity.  If 
the  supply  continues,  the  toxin  next  affects  the  nearest  sensory  appa- 
ratus ;  there  is  an  increase  in  the  reflexes,  but  only  when  the  affected 
portion  is  irritated.  In  the  further  course  of  the  poisoning  the  toxin 
as  it  ascends  continues  to  affect  more  and  more  motor  centres,  and 
also  the  neighboring  sensory  apparatus.  This  leads  to  spasm  of  all  the 
striated  muscles  and  general  reflex  tetanus. 

If  the  toxin  gets  into  the  blood  the  only  path  of  absorption  to  the 
central  nervous  system  is  still  by  way  of  the  motor-nerve  tracts.  There 
seems  to  be  no  other  direct  path,  as,  for  example,  by  means  of  the 
bloodvessels  supplying  the  central  nervous  system.  Even  after  intro- 


230  BACTERIA  PATHOGENIC  TO  MAN 

ducing  the  poison  into  the  subarachnoid  space,  owing  to  the  passage 
of  the  poison  into  the  blood,  there  is  a  general  poisoning  and  not  a 
cerebral  tetanus.  This  at  least  is  the  case  if  care  has  been  taken  during 
the  operation  to  avoid  injuring  the  brain  mechanically. 

If  now  we  follow  the  course  of  the  antitoxin  in  the  organism  as 
we  have  just  done  with  the  toxin,  we  find  that:  whereas  the  toxin 
passes  directly  to  the  nerve  paths,  the  antitoxin  when  injected  sub- 
cutaneously  is  entirely  taken  up  by  the  blood-vessels  by  means  of  the 
lymph- vessels.  From  the  blood  it  then  reaches  the  tissues,  with  the 
fluids  of  which  it  mixes. 

The  complete  absorption  of  a  given  quantity  of  antitoxin  adminis- 
tered subcutaneously  takes  place  rather  slowly.  In  his  animal  experi- 
ments Knorr  found  the  maximum  quantity  in  the  blood  only  after 
twenty-four  to  forty  hours.  From  that  time  on  the  amount  again 
steadily  decreased,  so  that  by  the  sixth  day  only  one-third  the  optimum 
quantity  was  present.  By  the  twelfth  day  only  one-fiftieth  and  at  the 
end  of  three  weeks  no  antitoxin  whatever  could  be  demonstrated. 

Naturally  the  time  during  which  these  changes  take  place  varies 
with  the  application,  the  conditions  of  absorption,  and  the  concentra- 
tion and  amount  of  the  preparation  injected.  When  injected  intra- 
venously the  antitoxin  very  quickly  passes  into  the  lymph.  Ransom 
was  able  to  demonstrate  it  in  the  thoracic  duct  of  a  dog  a  few  minutes 
after  intravenous  injection.  Neither  the  central  nervous  system  nor  the 
peripheral  nervous  tissue  take  up  any  antitoxin  from  the  blood.  Only 
after  very  massive  intravenous  doses  are  small  traces  found  in  the 
cerebrospinal  fluid.  From  this  it  is  at  once  clear  that  passively  and 
actively  immunized  animals  become  tetanic  if  the  poison  is  injected 
directly  into  the  central  nervous  system  or  into  a  peripheral  nerve. 
Antitoxin  injected  subdurally  also  passes  almost  entirely  over  into  the 
blood. 

A  rapid  and  plentiful  appearance  of  antitoxin  in  the  blood  is  depend- 
ent on  the  content  of  serum  in  antitoxin  units.  The  more  units,  the 
more  rapidly  will  the  blood  develop  a  high  content  of  antitoxin;  and 
the  higher  this  is  the  more  thoroughly  will  the  tissues  be  saturated  with 
the  antitoxin. 

From  the  foregoing  it  is  not  difficult  to  formulate  the  conditions 
under  which  an  antitoxin  introduced  into  the  organism  can  exert  its 
neutralizing  power  on  the  toxin.  We  see  that  the  poison  deposited  at 
any  given  place  takes  either  of  two  paths  to  the  central  nervous  system, 
one  a  direct  path  by  way  of  the  local  peripheral  nerves  and  the  other 
an  indirect  path  through  the  lymph  channels  and  blood  to  the  end 
plates  of  all  other  motor  nerves.  From  intact  bloodvessels  the  anti- 
toxin penetrates  neither  the  substance  of  the  peripheral  nerves  nor 
the  substance  of  the  central  nervous  system.  Hence,  only  that  portion 
can  be  neutralized  which  (a)  still  lies  unabsorbed  at  the  site  of  inocula- 
tion, or  (6)  which,  though  it  has  passed  into  the  blood,  has  not  yet  been 
taken  up  by  the  motor-nerve  endings.  A  curative  effect  can  therefore 
result  from  antitoxin  introduced  subcutaneously  or  intravenously  only 


TIIK  BACILLUS  AM)  Till-:  BACTERIOLOGY  OF  TETANUS      231 

so  long  as  a  fatal  dose  of  poison  has  not  been  taken  up  by  the  nerves. 
After  tli  is  has  occurred  an  action  of  the  antitoxin  can  only  then  be  looked 
for  when  it  is  injected  directly  into  the  nerve  substance. 

So  long  as  the  toxin  circulates  in  the  blood  it  is  neutralized  by  anti- 
toxin in  about  the  same  proportion  as  in  test-tube  experiments.  By 
means  of  intravenous  injections  of  antitoxin  Ransom  was  able  to  render 
the  blood  free  from  toxin  in  a  very  few  minutes.  According  to  Marie 
and  Morax  toxin  injected  into  the  muscles  is  already  demonstrable  in 
the  nerve  tissue  at  the  end  of  one  and  a  half  hours — i.  e.,  it  has  already 
entered  the  channel,  where  it  is  no  longer  reached  by  the  antitoxin. 
There  must,  however,  be  a  condition  or  locality  in  which  the  toxin  can 
still  be  neutralized  by  means  of  large  doses,  though  with  difficulty. 
This  is  indicated  among  other  experiments  by  some  older  researches 
of  Donitz.  This  observer  injected  various  rabbits  intravenously  each 
with  1  c.c.  of  a  toxin  solution  containing  twelve  fatal  doses.  Thereupon 
he  determined  the  dose  of  antitoxin  which,  when  intravenously  given, 
would  neutralize  this  poison  after  various  intervals  of  time.  The  anti- 
toxin was  of  such  a  strength  that  in  test-tube  experiments  1  c.c.  of  a 
1 : 2000  solution  just  neutralized  the  amount  of  toxin  employed.  He 
found  that  at  the  end  of  two  minutes  double  the  dose  required  in  vitro 
would  still  neutralize  the  poison;  at  the  end  of  four  minutes  about  four 
times  the  dose  was  required,  and  at  the  end  of  eight  minutes  ten  times. 
Wlien  one  hour  had  been  allowed  to  elapse  forty  times  the  original 
dose  just  sufficed  to  protect  the  animal  from  death,  but  not  from  sick- 
ness. In  order  to  explain  these  results,  the  correctness  of  which  has 
been  confirmed  bv  many  analogous  observations,  the  conception  "loose 
union  of  toxin"  has  been  introduced.  By  this  is  meant  a  state  of 
union  between  toxin  and  susceptible  cell  constituent  which  can  still 
be  disrupted  by  means  of  large  doses  of  antitoxin.  In  this  particular 
instance  we  do  not  need  to  make  use  of  this  conception,  for  the 
reason  that  the  tetanus  toxin  is  not  at  all  combined  during  the  first  hour, 
being  engaged  at  this  time  in  traversing  the  peripheral  nerve  paths. 
Personally,  I  should  regard  it  as  more  probable  that  the  interval  during 
which  the  toxin  can  still  be  neutralized,  though  with  difficulty,  corre- 
sponds to  that  time  during  the  passage  of  the  toxin  in  which  after  leav- 
ing the  capillaries  the  poison  is  held  up  in  the  fine  interstices  of  the 
connective  tissue  which  it  must  penetrate  before  it  can  be  taken  up  by 
the  nerves. 

Results  of  the  Antitoxin  Treatment  in  Tetanus. — Tetanus  is  a  com- 
paratively rare  disease  both  in  man  and  animals,  though  in  some 
localities  it  is  more  common  than  in  others.  In  New  York  City  there 
are  usually  fifteen  to  thirty  cases  following  every  Fourth  of  July.  Most 
of  them  are  caused  by"  infection  through  blank-cartridge  wounds. 
Recovery  sometimes  follows  from  the  ordinary  symptomatic  treatment 
or  without  treatment  at  all,  so  that  the  statistics  of  cures  of  the  disease 
by  the  injection  of  antitoxic  serum  must  be  very  carefully  sifted  before 
they  can  be  accepted  as  reliable.  Lambert,  who  a  few  years  ago 
made  an  exhaustive  study  of  tetanus,  states  that  in  a  total  of  114  cases 


232  BACTERIA  PATHOGENIC  TO  MAN 

of  this  disease  treated  with  antitoxin,  according  to  published  and  unpub- 
lished reports,  there  was  a  mortality  of  40.35  per  cent.  Of  these,  47 
were  acute  cases — that  is,  cases  with  an  incubation  period  of  eight  days 
or  less  and  with  rapid  onset,  or  cases  with  a  longer  period  of  incuba- 
tion, but  intensely  rapid  onset  of  symptoms;  of  these  the  mortality  was 
74.46  per  cent.  Of  the  chronic  type — those  with  an  incubation  period 
of  nine  days  or  more,  or  those  with  shorter  incubation  with  slow  onset 
—there  were  61  cases,  with  a  mortality  of  16.39  per  cent.  With  a  still 
larger  number  of  cases  the  results  indicate  that  with  tetanus  antitoxin 
about  20  per  cent,  better  results  are  obtained  than  without. 

Treatment  of  Tetanus  by  Antitoxin. — For  immunization,  10  c.c.  of  a 
serum  of  medium  strength  will  suffice  unless  the  danger  seems  great, 
when  the  injection  is  repeated  at  the  end  of  a  week.  For  treatment, 
it  is  well  to  begin  with  30  to  50  c.c.,  and  then,  according  to  the 
severity  of  the  case,  give  from  20  to  50  c.c.  each  day  until  the  symptoms 
abate.  In  the  gravest  cases  no  curative  effect  will  be  noticed  from  the 
serum.  The  first  injections  should  be  made  intravenously,  or  partly 
intravenously  and  partly  into  the  spinal  canal  through  lumbar  punc- 
ture. Later,  injections  should  be  made  subcutaneously  or  intravenously. 
Besides  these,  injections  should  be  made  into  all  the  nerve  trunks  leading 
from  the  infected  region.  These  injections  should  be  made  as  near  the 
trunk  as  possible  and  distend  the  nerve  so  as  to  partly  neutralize  and 
partly  mechanically  interrupt  the  passage  of  toxin  to  the  cord  or  brain. 
In  New  York  City  Rogers  has  had  good  results  by  following  these 
cases.  He  has  also  injected  the  antitoxin  directly  into  the  spinal  cord. 
The  method  of  injecting  from  3  to  15  c.c.  of  antitoxic  serum  into  the  lateral 
ventricles  has  not,  in  the  writer's  opinion,  shown  itself  to  be  equal  to  the 
intravenous  and  intraspinal  and  intraneural  methods.  No  bad  results 
have  followed  the  injections  when  the  serum  was  sterile  and  the  opera- 
tion was  performed  aseptically;  but  several  brain  abscesses  have  already 
followed  the  intracerebral  injections. 

The  striking  results  which  have  been  obtained,  particularly  in  veter- 
inary practice,  with  the  prophylactic  injection  of  tetanus  antitoxin, 
would  seem  to  warrant  the  treating  of  patients  with  immunizing  doses 
of  serum — at  least,  in  neighborhoods  where  tetanus  is  not  uncommon— 
when  the  lacerated  and  dirty  condition  of  their  wounds  may  indicate 
the  possibility  of  a  tetanus  infection. 

Differential  Diagnosis. — The  differential  diagnosis  of  the  bacillus  of 
tetanus  is,  generally  speaking,  not  difficult,  inasmuch  as  animal  inocu- 
lation affords  a  sure  test  of  the  specific  organism.  No  other  micro- 
organism known  produces  similar  effects  to  the  tetanus  bacillus,  nor 
is  any  other  neutralized  by  tetanus  antitoxin.  The  other  character- 
istics also  of  this  bacillus  are  usually  distinctive,  though  microscopic 
examination  alone  cannot  be  depended  on  to  make  a  differential  diag- 
nosis. Difficulty  arises  when  other  anaerobic  or  aerobic  bacilli,  almost 
morphologically  identical  with  the  tetanus  bacillus,  are  encountered 
which  are  non-pathogenic,  such  as  the  bacillus  pseudotetanicus  anaero- . 
bins,  already  mentioned,  and  the  bacillus  pseudotetanicus  aerobius.  It 


THE  BACILLUS  AND  THE  BACTERIOLOGY  OF  TETANUS      233 

it  possible,  however,  that  both  these  bacilli,  when  characteristic  in  cul- 
tures, are  only  varieties  of  the  tetanus  bacillus,  which,  under  unfavor- 
able conditions  of  growth,  have  lost  their  virulence.  These  non-virulent 
types  do  not,  as  a  rule,  have  spores  absolutely  at  their  ends,  and  the 
spores  themselves  are  usually  more  ovoid  than  those  in  the  true  tetanus 
bacilli. 

METHODS  OF  EXAMINATION  IN  A  CASE  OF  TETANUS,  (a)  Micro- 
scopic.— From  every  wound  or  point  of  suppuration  film  preparations 
should  be  made  and  stained  with  the  usual  dyes.  The  typical  spore- 
bearing  forms  are  looked  for,  but  are  usually  not  found.  At  the  same 
time  other  bacteria  are  noted  if  present. 

(6)  Cultures. — Bits  of  tissue,  pus,  cartridge  wads,  etc.,  are  collected 
and  dropped  into  glucose  bouillon  contained  in  small  flasks  or  tubes. 
This  bouillon  should  be  slightly  alkaline,  be  free  from  oxygen,  and 
protected  from  oxygen.  A  simple  way  is  to  cover  the  bouillon  with 
liquid  or  semisolid  paraffin,  and  thus  boil  it.  Cultures  placed  in  such 
protected  bouillon  grow  readily. 

(c)  Inoculation. — Material  from  the  wound  is  inoculated  into  mice 
or  guinea-pigs  subcutaneously. 


CHAPTER  XIX. 

THE  COLON  BACILLUS  GROUP,  PARACOLON,  PARATYPHOID 
DYSENTERY  AND  PARADYSENTERY  BACILLI. 

The  Colon  Bacillus  Group. 

THE  first  organism  of  this  group  was  described  by  Emmerich  (1885), 
who  obtained  it  from  the  blood,  various  organs,  and  intestinal  dis- 
charges of  cholera  patients  at  Naples.  Similar  organisms  were  after- 
ward found  by  Escherich  (1886)  in  the  feces  of  healthy,  milk-fed  infants. 
It  has  since  been  demonstrated  that  members  of  the  colon  group  are 
normal  inhabitants  of  the  intestines  of  man  and  of  most  of  the  lower 
animals. 

Morphology. — The  bacillus  coli  varies  considerably  in  its  morphology, 
according  to  the  sources  and  the  culture  media  from  which  it  is 
obtained.  The  typical  form  (Fig.  85)  is  that  of  short  rods  with 
rounded  ends,  from  OA/J.  to  0.7//  in  diameter  by  1,«  to  3,«  in  length; 
sometimes,  especially  where  the  culture  media  are  not  suitable  for  their 
growth,  the  rods  are  so  short  as  to  be  almost  spherical,  resembling 
micrococci  in  appearance,  and,  again,  they  are  somewhat  oval  in  form 
or  are  seen  as  threads  of  6,u  or  more  in  length.  The  various  forms 
may  often  be  associated  in  the  same  culture.  The  bacilli  may  occur 
as  single  cells  or  as  pairs  joined  end-to-end,  rarely  as  short  chains. 
Capsules,  though  present,  are  not  shown  by  the  ordinary  methods. 

FLAGELLA. — Upon  some  varieties  seven  or  eight  flagella  have  been 
demonstrated,  upon  others  none.  The  flagella  are  shorter  and  more 
delicate  than  those  characteristic  of  the  typhoid  bacilli.  There  is 
nothing  in  the  morphology  of  this  bacillus  sufficiently  characteristic  for 
its  identification,  for  in  this  respect  it  simulates  many  other  organisms. 

Staining. — The  colon  bacillus  stains  readily  with  the  ordinary  aniline 
colors;  it  is  always  decolorized  by  Gram's  method. 

Biology. — It  is  an  aerobic,  facultative  anaerobic,  non-liquefying  bacil- 
lus. It  develops  best  at  37°  C.,  but  grows  well  at  20°  C.  and  slowly  at 
10°  C.  It  is  usually  motile,  but  the  movements  in  some  of  the  cultures 
are  so  sluggish  that  a  positive  opinion  is  often  difficult.  In  fresh  cultures 
frequently,  only  one  or  two  individuals  out  of  many  show  motility. 

Cultivation. — The  colon  bacillus  develops  well  on  all  the  usual  cul- 
ture media.  Its  growth  on  them  is  usually  more  abundant  than  that 
of  the  typhoid  bacillus  or  the  dysentery  bacillus,  but  the  difference 
is  not  sufficient  for  a  differential  diagnosis.  Although  it  grows  best 
aerobically,  yet  it  grows  well  anaerobically,  especially  in  media  contain- 
ing fermentable  sugars. 


THE  COLON  BACILLUS  GROUP  235 

GELATIN. — In  gelatin  plates  colonies  are  developed  in  from  eighteen 
to  thirty-six  hours.  They  resemble  greatly  the  colonies  of  the  typhoid 
bacillus,  except  that  many  of  them  are  somewhat  larger  and  more 
opaque.  When  located  in  the  depths  of  the  gelatin  and  examined  by 
a  low-power  lens  they  are  at  first  seen  to  be  finely  granular,  almost 
homogeneous,  and  of  a  pale  yellowish  to  brownish  color;  later  they 
become  larger,  denser,  darker,  and  more  coarsely  granular.  In  shape 
they  may  be  round,  oval,  or  whetstone-like.  The  superficial  colonies 
appear  as  small,  dry,  irregular,  flat,  blue-white  points,  that  are  com- 
monly somewhat  dentated  at  the  margins.  When  the  gelatin  is  not 
firm  the  margins  of  many  colonies  are  broken  by  outgrowths,  which 
are  rather  characteristic  of  colon  bacilli.  Under  the  same  conditions 
the  colonies  of  typhoid  bacilli  usually  show  thread-like  outgrowths. 


FIG.  85 


Colon  bacilli.    Twenty-four-hour  agar  culture.    X  1100  diameters. 

In  stab  cultures  on  gelatin  the  growth  usually  takes  the  form  of  a 
nail  with  a  flattened  head,  the  surface  extension  generally  reaching 
out  rapidly  to  the  sides  of  the  tube. 

NUTRIENT  AGAR. — In  plate  cultures :  Surface  colonies  mostly  circular, 
finely  granular,  and  rather  opaque.  The  deep  colonies  are  apt  to  have 
protuberances.  There  are  no  marked  differences  between  colonies  of 
colon  and  typhoid  bacilli.  In  streak  cultures  an  abundant,  soft,  white 
layer  is  quickly  developed,  but  the  growth  is  not  characteristic. 

'BOUILLON.— In  bouillon  the  bacillus  coli  produces  diffuse  clouding 
with  sedimentation;  in  some  cultures  a  tendency  to  pellicle  formation 
on  the  surface  is  occasionally  seen. 

POTATO. — On  potato  the  growth  is  rapid  and  abundant,  appearing 
after  twenty-four  to  thirty-six  hours  in  the  incubator  as  a  yellowish- 
brown  to  dark  cream-colored  deposit  covering  the  greater  part  of  the 
surface.  But  there  are  considerable  variations  from  the  typical  growth 
on  potato;  there  may  be  no  visible  growth  at  all,  or  it  may  be  scanty  and 
of  a  white  color.  These  variations  are  due  at  times  to  the  bacillus, 
but  more  often  to  variations  in  the  potato. 


236  BACTERIA  PATHOGENIC  TO  MAN 

COAGULATED  SERUM. — The  serum  is  not  liquefied.  On  this  medium 
grayish,  uncharacteristic  colonies  are  developed. 

MILK  coagulates  in  from  four  to  ten  days  at  20°  C.,  and  in  from  one 
to  four  days  at  37°  C.  Coagulation  is  aided  by  the  addition  of  pep- 
tone; it  is  prevented  by  constant  addition  of  alkalies.  The  acids 
formed  are  lactic,  acetic,  formic,  and  succinic  acids.  Coagulation  is 
due  principally  but  not  altogether  to  acids.  A  ferment  is  produced 
which  is  capable  of  causing  coagulation  in  the  presence  of  lime 
salts,  especially  in  acid  solutions.  It  is  evident  also  that  the  nature 
of  this  coagulation  is  more  closely  related  to  coagulation  fermentation 
than  to  simple  acid  fermentation,  from  the  fact  that  colon  coagulation 
forms  a  compact  mass  which  is  difficultly  soluble  in  alkalies,  and 
contains  much  insoluble  residue;  and  further,  that  in  serum  coagulated 
by  colon  bacilli  a  proteid  body  corresponding  to  fermentation  albumin 
is  found. 

In  addition  to  albumose  it  has  been  shown  that  milk  serum,  after 
colon  coagulation,  contains  a  substance  possessing  the  reaction  of  pep- 
tone, which  is  not  contained  in  the  original  milk.  Similar  albumin 
cleavage  products  are  also  formed  in  cultures  of  bacillus  coli  in  sugar- 
free  ascitic  fluid;  it  cannot  be  assumed,  therefore,  that  colon  acidification 
of  milk,  as  such,  produces  this  proteolysis.  The  colon  bacilli  also  differ 
from  typhoid  bacilli  in  that  their  action  on  milk-sugar  is  more  intense, 
and,  since  they  can  grow  in  greater  acidity,  more  lasting.  The  dif- 
ference, therefore,  is  quantitative  rather  than  qualitative. 

Chemical  Activities.  BEHAVIOR  TOWARD  CARBOHYDRATES. — In  cul- 
tures of  colon  bacilli  many  carbohydrates,  especially  sugars,  become 
fermented.  Among  these  are: 

Hexoses,  CfiH12O6  (glucose,  mannose,  fructose,  galactose). 

Pentoses,  C5H10O5  (arabinose,  xylose). 

Binoses,  C12H22Olt   (saccharose,  lactose,  maltose,  melitose). 
Also  the  higher  alcohols: 

Hexatomic  alcohol  (mannit,  dulcit,  sorbit). 

Pentatomic  alcohol  (erytherit). 

Triatomic  alcohol  (glycerin). 

The  varieties  included  under  colon  bacilli  vary  in  their  action  on 
some  of  the  sugars;  some  ferment  cane-sugar,  others  do  not.  All  fer- 
ment glucose.  For  fermentation  they  require  nitrogenous  substances 
which  can  be  utilized  as  food  by  the  bacteria,  a  suitable  temperature 
(best  at  30°  to  35°  C.),  absence  of  deleterious  substances. 

The  products  derived  from  the  splitting  of  the  carbohydrates  are 
as  follows:  From  glucose,  saccharose,  arabinose,  and  galactose  there 
is  mainly  produced  /cpi>o-lactic  acid  along  with  5  to  25  per  cent  of 
dextro-lactic  acid;  mannit,  on  the  contrary,  yields  only  /«i>o-lactic  acid. 
The  lactic  acid  formed,  however,  corresponds  to  only  one-half  the 
quantity  of  sugar  present.  The  by-products  are :  gaseous  compounds, 
alcohol,  succinic,  acetic,  and  formic  acids.  The  most  important  fermen- 
tation products,  both  qualitatively  and  quantitatively,  are  produced 
from  grape-sugar,  probably,  according  to  the  following  reaction: 


THE  COLON  BACILLUS  GROUP  237 

2C6H12O6  -f  H20  =  2C3H603  +  CH3COOH  +  C2H5COOH  -f  2CO,  +  2H2 
Grape-sugar.       Water.       Lactic  acid.        Acetic  acid.          Ethyl  alcohol.     Carbonic    Hydrogen. 

acid. 

ACID  PRODUCTION  FROM  SUGARS  BY  COLON  BACILLI. — When  suffi- 
cient sugar  is  present  the  amount  of  acid  produced  is  quite  uniform. 
In  many  it  proceeds  until  the  acidity  is  sufficient  in  quantity  to  stop 
the  growth  of  the  bacilli.  In  milk  this  acidity  becomes  about  12  per 
cent,  f,  acid  to  phenolphthalein.  There  are  reasons  to  think  that  lactic 
acid  is  first  produced  and  that  from  this  other  acids  and  products  de- 
velop. Under  aerobic  conditions  lactic  is  produced  in  excess  of  acetic 
acid,  while  in  the  absence  of  oxygen  the  reverse  is  apt  to  be  true. 

GAS  PRODUCTION. — When  colon  bacilli  are  grown  in  a  solution  of 
glucose,  CO2  and  H2  are  produced,  1CO2  to  1H2  up  to  1CO2  to  3H2. 
Anaerobic  conditions  aid  gas  formation.  Some  colon  varieties  produce 
gas  from  no  sugars  and  some  from  a  few  only.  Nearly  all  produce  gas 
from  glucose,  and  about  60  per  cent,  of  varieties  produce  gas  from 
milk-sugar.  Very  slight  traces  of  gases  other  than  H  and  CO2  are  pro- 
duced. The  amount  of  gas  varies  in  different  varieties;  the  closed  arm 
of  the  tube  half -filled,  and  the  II  and  CO2  in  the  proportion  2  to  1, 
is  the  characteristic  type.  It  is  also  true  of  Gartner's  B.  enteritidis.  In 
another  type  the  whole  of  the  closed  arm  is  filled, — H2:CO2=1:2  or  3. 
This  type  is  usually  called  bacterium  cloacae.  In  a  third  type  the  arm 
is  nearly  filled, — H2:CO2=1:1.  This  type  is  the  bacterium  lacticum 
aerogenes. 

Concerning  the  gas  and  acid  produced  the  following  rules  have  been 
established : 

1.  The  quantity  produced  varies  with  the  amount  of  bacteria  and 
their  activity  as  well  as  with  the  type  of  organism. 

2.  It  depends  upon  the  living  organisms. 

3.  The  products  have  a  less  heat  value  than  the  sugar  from  which 
they  were  formed. 

4.  The  fermentation  is  not  a  simple  hydrolytic  action,  but  one  in 
which  combinations  between  the  C  and  O  atoms  are  sundered  and 
formed.    This  is  not  an  oxidation  process,  but  a  change  through  break- 
ing down — that  is,  a  true  decomposition.    What  oxidation  takes  place  is 
chiefly  due  to  the  oxygen  liberated  from  splitting  the  sugar  molecules. 

USE  OF  SUGAR  LITMUS  AGARS  TO  DIFFERENTIATE  BETWEEN  COLON  AND 

TYPHOID   BACILLI. 

Color  of  media  after  24  hours'  growth  of  culture. 
List  of  sugars. 

Colon  bacillus.  Typhoid  bacillus. 

Grape-sugar        .         .         .  Ked.  Red. 

Saccharose  ....  Bed.  Red. 

Mannite      ....  Red.  Red. 

Cane-sugar  .         .         .  Blue.  Blue. 

Maltose Red  or  moderately  red.  Red  or  pink. 

Milk-sugar  .         .         .  Red.  Blue. 

Dextrin       ....  Blue.  Violet  blue. 
To  bouillon  used  for  acid  and  gas  formation  no  sodium  hydrale  or  carbonate  should 
be  added. 


238  BACTERIA  PATHOGENIC  TO  MAN 

EFFECT  OF  COLON  BACILLI  IN  NITROGENOUS  COMPOUNDS.  —  Colon 
bacilli  do  not  appreciably  liquefy  gelatin  nor  peptonize  any  albumins. 
They  do,  however,  break  down  some  of  the  higher  nitrogenous  com- 
pounds into  smaller  atom  groups.  The  first  noted  of  these  compounds 

was  indol,  C6H4v      rr  /  CH.    This  is  one  of  the  most  important  products 


of  colon  activity,  although  a  few  varieties  lack  it.  (Witte's  peptone  solu- 
tion is  used  as  test.)  Sugars  interfere  with  indol  production,  as  also  does 
the  absence  of  oxygen.  The  maximum  amount  of  indol  is  present 
about  the  tenth  day.  The  test  is  carried  out  by  adding  1  c.c.  of  0.02 
per  cent,  potassium  nitrite  to  10  c.c.  of  culture  fluid,  and  then  some 
concentrated  sulphuric  acid.  To  prevent  confusion  with  other  colors  a 
little  amyl  alcohol  is  added  and  shaken.  In  the  intestinal  canal  in  health 
very  little  indol  appears  to  be  produced  by  colon  bacilli.  Sulphuretted 
hydrogen  is  liberated  from  sugar-free  fermentable  proteid  substances. 
Mercaptan  and  sometimes  skatol  have  been  noted  in  peptone  solution 
cultures.  The  colon  bacillus  liquefies  minute  quantities  of  gelatin,  but 
so  little  as  to  be  inappreciable. 

The  colon  bacillus  can  make  use  of  simpler  nitrogen  compounds 
than  the  typhoid  bacillus,  and  can  grow  well  in  media  such  as  that  of 
Uschinsky.  In  urine  some  colon  cultures  produce  a  slight  fermentation, 
yielding  ammonium  carbonate.  By  some  this  is  believed  not  to  be  due 
to  urea  fermentation.  Lactose-litmus-urine  agar,  after  twenty-four 
hours'  acidity,  often  becomes  alkaline. 

In  media  containing  fermentable  sugars  and  proteid  substances 
simultaneous  action  takes  place  on  both  with  the  production  of  both 
alkalies  and  acids. 

Effect  on  Fats.  —  No  action  has  been  noted. 

Reduction  Processes.  REDUCTION  OF  PIGMENTS.  —  The  action  on 
certain  pigments  which  are  reduced  to  colorless  products  and  interme- 
diate colors  is  more  vigorous  than  that  of  typhoid  bacilli.  This  effect 
occurs  in  litmus  bouillon  in  the  closed  arm  of  the  tube,  in  bouillon 
and  agar  (not  in  gelatin),  indigo-sodium-sulphate-methyl  blue,  in  sugar 
media,  etc. 

REDUCTION  OF  INORGANIC  SALTS.  —  From  nitrates  coli  under  certain 
conditions  produce  nitrites  and  from  them  free  nitrogen  as  follows: 
2NaN03  +  H2O  =  2NaOH  +  N2  +  O5. 

The  development  of  pigments  (brownish,  greenish,  and  yellowish), 
odorous  substances,  and  toxins  has  been  noted,  especially  on  potato. 
Besides  ammonia  and  the  fatty  and  oxy-fatty  acids  other  bad  -smelling 
products,  but  little  understood,  are  formed  at  times. 

Toxins.  —  The  bodies  of  dead  colon  bacilli  contain  pyogenic  substances, 
and  others  which,  injected  into  the  circulation,  produce  paralysis  of  the 
striped  muscle  fibres,  convulsions,  coma,  and  death.  Extracts  from 
some  cultures  produce  irritation  of  the  mucous  membranes  of  the  large 
intestines  with  dysenteric  symptoms. 

Growth  with  Other  Bacteria.  —  Colon  bacilli  grown  with  typhoid  bacilli 
are  hindered  in  'their  gas  production  and  indol  formation,  while  the 


THE  COLON  BACILLUS  GROUP  239 

latter  soon  die  out.  (Typhoid  bacilli  usually  die  out  in  the  filtrate  of 
colon  culture.)  Colon  varieties  differ  as  to  whether  they  increase  or 
not  in  the  old  culture  fluids  of  other  broth  colon  cultures.  The  colon 
bacilli  act  antagonistically  to  many  of  the  proteolytic  bacteria  in  the 
intestinal  tract,  and  so  inhibit  alkaline  putrefaction  by  them.  In  milk 
the  same  antagonism  exists,  probably  because  of  the  acidity  caused 
by  the  colon  growth. 

Reaction  to  Temperature. — Growth  takes  place  best  at  37°  C.;  the 
range  is  from  5°  to  45°  C.  Colon  bacilli  are  killed  at  60°  C.  in  from 
five  to  fifteen  minutes.  Frozen  in  ice  a  large  proportion  die,  but  some 
resist  for  six  months.  In  liquid  air  95  per  cent,  are  killed  in  two 
hours. 

Resistance  to  Drying. — The  colon  bacillus  is  quite  resistant.  Simple 
drying  destroys  the  majority  of  organisms  dried  at  any  one  time,  but 
some  bacilli  of  the  number  dried  may  remain  alive,  especially  when 
held  in  the  texture  of  threads,  for  five  to  six  months,  or  all  may  die  in 
forty-eight  hours. 

Effect  of  Light. — In  water  exposed  to  direct  sunlight  colon  bacilli 
are  killed  in  from  one  to  six  hours.  In  diffuse  light  a  moderate  effect 
only  is  produced.  They  are  fairly  resistant  to  Roentgen  rays,  quite 
so  to  the  electric  current. 

Effect  of  Acids. — The  colon  bacilli  grow  in  a  wider  range  of  acids 
and  alkalies  than  most  other  bacteria.  They  develop  in  from  0.2  to  0.4 
per  cent,  of  mineral  acids  in  from  0.3  to  0.45  per  cent,  of  vegetable 
acids,  and  in  from  0.1  to  0.2  per  cent,  of  alkalies. 

To  most  antiseptics  they  are  resistant,  growing  feebly  even  in  1 : 5000 
formaldehyde.  They  grow  in  from  6  to  8  per  cent,  of  salt  and  live  in 
concentrated  solutions. 

Effect  of  Animal  Fluids  and  Juices. — Gastric  juice  kills  colon  bacilli 
when  not  protected  or  too  greatly  diluted  by  food.  They  grow  in  bile 
and  in  the  intestinal  juices.  Fresh  rabbit  and  dog  blood  kills  colon 
bacilli,  as  also  but  more  quickly  typhoid  bacilli.  Calf  and  horse  serum 
are  said  to  be  inactive. 

Virulence  of  Colon  Bacilli.  Pathogenic  Properties  in  Animals. — The 
virulence  varies  with  the  culture  and  the  time  since  its  recovery  from 
the  intestines.  Other  things  being  equal,  it  is  more  virulent  from  an 
intestinal  inflammation.  From  severe  diarrhoea  the  colon  bacilli  in 
0.25  c.c.  bouillon  culture  may  kill  guinea-pigs  if  given  intraperitoneally, 
while  from  healthy  bowel  2.5  c.c.  are  usually  required.  Smaller  amounts 
are  fatal  to  white  mice. 

INCREASE  OF  VIRULENCE  OUTSIDE  THE  BODY. — It  has  been  found 
by  several  observers  that  in  fermenting  fecal  matter  a  marked  increased 
virulence  takes  place,  so  that  infection  is  produced  when  received  by 
man.  Some  investigators  claim  that  by  growing  the  colon  bacilli  with 
typhoid  bacilli  and  with  the  pyogenic  cocci  increase  of  virulence  occurs. 
This  is  improbable  outside  the  body.  The  lesions  following  injection 
with  colon  bacilli  do  not  differ  from  those  produced  by  the  typhoid 
bacilli. 


240  BACTERIA  PATHOGENIC  TO  MAN 

Pathogenesis. — The  lesions  present  in  intestinal  infection  are  those 
of  enteritis ;  the  duodenum  and  jejunum  are  found  to  contain  fluid,  the 
spleen  is  somewhat  enlarged,  and  there  is  marked  hyperaemia  and  ecchy- 
mosis  of  the  small  intestines,  together  with  swelling  of  Peyer's  patches. 

Intraperitoneal  and  intravenous  inoculation  of  guinea-pigs  and  rab- 
bits may  also  produce  death,  which,  when  it  follows,  usually  takes 
place  within  the  first  forty-eight  hours,  accompanied  by  a  decided  fall 
of  temperature,  the  symptoms  of  enteritis,  diarrhoea,  etc.,  and  finally 
fibropurulent  peritonitis. 

When  subcutaneous  inoculations  of  mice  and  guinea-pigs  are  made 
it  requires  the  introduction  of  much  larger  quantities  of  the  cultures 
to  produce  infection;  in  rabbits  this  is  followed  only  by  abscess  forma- 
tion at  the  point  of  inoculation.  Dogs  and  cats  are  similarly  affected. 

Albarran  and  Halle*  have  caused  cystitis  and  pyelonephritis  by  direct 
injections  into  the  bladder  and  ureters,  the  urine  being  artificially  sup- 
pressed; Chassin  and  Roger  produced  angiocholitis  and  abscess  of  the 
liver  in  the  same  way.  Akermann  produced  osteomyelitis  in  young 
rabbits  by  intravenous  injections  of  cultures. 

From  experiments  on  animals  it  would,  therefore,  appear  that  the 
true  explanation  of  the  pathogenesis  of  the  colon  bacillus  is  undoubt- 
edly to  be  found  in  the  toxic  effects  of  the  chemical  substance  and 
products  of  the  cells. 

Immunization. — Immunization  against  colon  bacillus  infection  is  pro- 
duced in  the  same  way  and  to  the  same  degree  as  in  typhoid  bacillus 
infection.  The  serum  from  a  horse  receiving  a  number  of  different 
strains  is  required,  if  protection  from  all  members  of  the  colon  group 
is  desired.  The  serum  is  not  at  present  employed  therapeutically. 

Occurrence  in  Man  and  Animals  during  Health. — The  bacillus  coli 
communis  is  a  common  inhabitant  of  the  intestinal  canal  in  man  and 
in  almost  all  domestic  animals.  It  is  also  found  at  times  in  wild  animals 
and  appears  at  times  to  develop  in  fishes. 

Occurrence  Outside  of  the  Intestines. — Colon  bacilli  are  found  wherever 
human  or  animal  feces  are  carried.  In  water  a  very  few  colon  bacilli, 
less  than  one  to  each  10  c.c.,  are  not  sufficient  to  give  rise  to  the  sus- 
picion that  it  is  contaminated.  Even  1  per  c.c.  does  not  show  human 
contamination,  since  such  a  number  could  equally  well  come  from  manure 
spread  on  fields.  Colon  bacilli  are  apt  to  be  found  in  everything  which 
comes  in  contact  with  man  or  animals,  or  dust  where  they  have  been. 

Variation  in  Morphological  and  Biological  Characters. — By  subjecting 
them  to  higher  than  normal  temperature,  to  long  growth  in  old  culture 
fluids,  to  the  action  of  weak  antiseptics,  and  to  the  passage  through 
animals,  colon  bacilli  have  been  changed  so  as  to  lose  the  power  to  make 
indol  and  ferment  sugars  with  gas  production.  They  have  not,  however, 
been  made  to  approach  typhoid  bacilli  in  their  agglutination  charac- 
teristics or  in  their  motility.  The  change  has  simply  been  to  weaken  the 
characteristics  typical  of  colon,  and  this  gives  them  appearances  similar 
to  typhoid  bacilli,  but  in  parasitic  ways  they  are  still  quite  different. 

To  the  colon  group  the  following  characteristics  are  essential :    short 


THE  COLON  BACILLUS  GROUP 


241 


rod  form;  relatively  good  growth  on  media;  decolorization  by  Gram; 
the  lack  of  appreciable  liquefaction  of  gelatin;  the  facultative  properties; 
indol  formation;  sugar  fermentation;  milk  coagulation;  motility;  patho- 
genic properties  in  animals  to  greater  or  less  extent.  The  saprophytic 
life  of  the  colon  bacillus  leads  to  gradual  variations  according  to  con- 
ditions. 

Association  with  Other  Bacteria  in  the  Intestines. — In  infant  stools 
we  usually  find  in  ordinary  cultures  only  bacterium  coli  and  bacterium 
lactis  aerogenes.  Both  fail  to  ferment  albumin,  and  both  markedly 
ferment  carbohydrates.  The  bacterium  lactis  aerogenes  is  most  abundant 
in  the  upper  intestinal  tract,  the  colon  bacillus  in  the  lower  intestines. 
The  former  appears  to  feed  on  the  carbohydrates,  the  latter  on  sub- 
stances secreted  by  the  intestinal  mucous  membranes.  Only  about 
10  per  cent,  of  the  bacteria  seen  under  the  microscope  appear  as  colonies, 
and  whereas  in  infant  stools  the  majority  of  the  bacteria  are  frequently 
Gram  positive,  the  larger  number  of  the  colonies  are  composed  of 
Gram-negative  bacteria.  Some  of  the  Gram-positive  bacteria  are 
anaerobic;  others  fail  to  grow  on  ordinary  culture  media.  These  con- 
ditions, the  normal  presence  of  colon  bacilli  and  the  tendency  of  other 
bacteria  not  to  grow  in  culture  media,  make  the  greatest  care  neces- 
sary in  weighing  conclusions  as  to  the  pathogenic  significance  of  colon 
bacilli  in  disease.  Further,  although  most  colon  bacilli  seem  alike  in 
the  cruder  methods  formerly  employed,  it  is  now  found  that  there  are 
many  differences  in  their  action  upon  carbohydrates  and  in  their  agglu- 
tination affinities.  There  has  thus  come  to  be  a  group  of  colon  bacilli 
rather  than  a  colon  bacillus.  Escherich  restricts  the  name  to  bacilli 
having  the  characteristics  of  those  existing  in  the  intestines  of  normal 
nursing  infants. 

The  following  schematic  table  illustrates  some  of  the  characteristics 
of  members  of  the  colon  group  and  of  the  typhoid  bacilli : 


Bacilli  of  colon  group 


Typhoid  bacilli .    .  <  . 


Motility. 


D         — 

E 

-f 


Lactose 
fermen- 
tation. 


Milk         Indol  \  Growth 

coagula-        pro-       Flagellaj       on 
tion .       duction.  potato. 


+  -f 


- 


Growth  on 

Uchinsky 

media. 


The  Colon  Bacillus  under  Physiological  and  Pathological  Conditions. — 
In  the  breast-fed  infant  within  a  few  hours  or  days  after  birth  one  or  two 
varieties  of  typical  colon  bacilli  are  found  in  the  colon,  and  these  bacilli 
form  the  great  majority  of  all  the  bacteria  present  which  grow  in  media. 
The  bacilli  in  one  infant's  intestines  usually  all  agglutinate  with  the 
same  serum,  but  those  from  different  infants  vary.  The  bacilli  find 

16 


242  BACTERIA  PATHOGENIC  TO  MAN 

their  way  through  the  food  or  from  the  anus  upward.  In  the  small 
intestines  the  bacterium  lactis  aerogenes  is  most  prevalent,  while  in  the 
csecum  and  below  the  colon  types  predominate. 

In  healthy  intestines  a  function  of  colon  bacilli  is  supposed  to  be  to- 
prevent,  through  acids  and  other  products  formed,  the  development  of 
putrefactive  bacteria.  In  normal  intestines  with  intact  mucous  mem- 
branes the  toxic  products  formed  by  the  colon  bacilli  are  absorbed  but 
little  or  not  at  all,  and  the  bacilli  themselves  are  prevented  from  invad- 
ing the  tissues  by  the  epithelial  layer  and  the  bactericidal  properties  of 
the  body  fluids.  Possibly  there  is  an  acquired  immunity  to  the  colon 
varieties  which  have  long  inhabited  the  intestines. 

Behavior  during  Diarrhoea. — In  diarrhoea  we  find  increased  peristalsis, 
less  absorption  of  foodstuff,  increased  and  changed  intestinal  secre- 
tions. Tissier  observed  that  under  treatment  with  cathartics  the  colon 
varieties  increased,  while  the  anaerobic  forms  are  inhibited.  In  diarrhoea 
the  same  conditions  are  active,  inhibiting  causes  are  lessened,  and 
increased  mucus  and  serum  are  poured  out  into  the  canal.  This  is 
notably  seen  in  typhoid  fever.  In  diarrhoea,  although  the  common 
colon  varieties  are  met  with,  there  is  usually  seen  a  difference  in  that 
uncommon  varieties  and  more  typhoid-like  bacteria  are  also  found. 
Much  more  investigation  is  needed  on  this  complex  subject  of  varia- 
tion in  types  between  health  and  disease. 

Passage  of  Colon  Bacilli  through  the  Walls  of  the  Intestines  Just  Before 
and  After  Death. — The  colon  bacilli  tend  to  pass  through  the  intestinal 
walls  shortly  after  death  in  those  who  have  suffered  from  chronic  ill- 
ness. In  chronic  disease  the  resistance  of  the  tissues  is  lessened  or 
removed,  and  if  to  this  is  added  ulceration  or  loss  of  epithelium  in 
the  lining  of  the  intestines  the  entrance  of  the  bacteria  is  still  more 
favored.  The  temperature  at  which  the  body  is  kept  after  death  deter- 
mines often  whether  a  general  post-mortem  invasion  takes  place.  The 
passage  of  the  colon  bacilli  before  death  rarely  occurs  unless  some 
lesions  of  the  intestines  are  present. 

Varieties  of  Colon  Bacilli  as  Disease  Producers. — The  colon  bacillus 
was  at  first  regarded  purely  as  a  saprophyte.  Later,  through  not  realiz- 
ing the  post-mortem  invasion  and  the  great  ease  of  growth  of  the 
colon  bacillus  on  ordinary  media,  the  other  extreme  was  taken  of  attrib- 
uting too  much  to  it.  By  means  of  specific  sera  and  more  careful  study 
we  are  now  coming  to  a  clearer  view  of  the  action  of  this  micro-organism. 
The  colon  varieties  met  with  differ  considerably,  and  we  now  speak  of 
the  colon  group  rather  than  of  the  bacterium  coli  communis. 

It  is  best  to  try  and  separate  the  cases  which  come  from  varieties  of 
the  colon  bacillus  normally  present  in  the  intestines  from  those  derived 
from  outside  infection.  In  this  latter  class  the  bacilli  again  vary  from 
the  atypical  varieties  to  those  classed  out  of  the  group,  such  as  the  para- 
colon  bacilli,  the  dysentery  bacillus,  the  bacilli  from  meat  and  food 
infections,  etc.  These  bacilli  are  considered  under  their  special  headings. 

The  colon  bacilli  previously  in  the  intestines  can,  either  by  an  in- 
crease in  virulence  in  them  or  by  a  lowered  resistance  in  the  person, 


THE  COLOX  BACILLUS  GROUP  243 

invade  the  tissues  in  which  their  toxins  act,  causing  injury  to  the  intes- 
tinal tract.  Thus  in  the  case  of  ulceration  in  typhoid  fever  the  colon 
bacilli  enter  the  blood,  or  in  perforation  produce  peritonitis.  In  dying 
conditions  they  at  times  pass  through  the  intact  mucous  lining.  In 
the  gall-bladder  or  urinary  tract  the  spread  of  bacilli  from  the  intestines 
may  cause  disease.  The  virulence  of  the  colon  bacillus  in  endogenous 
infection  is  usually  somewhat  increased  over  that  which  it  possessed 
when  it  was  latent  in  the  intestine.  The  specific  serum  reaction  in  the 
body  is  a  sign  of  infection,  but  great  care  has  to  be  observed  in  deciding 
that  it  is  present,  as  group  agglutinins  also  occur.  Up  to  the  present 
time  it  is  very  difficult  to  state  in  any  colon  infection  whether  the 
bacilli  were  previously  present  in  the  intestines  or  were  derived  from 
outside. 

Colon  Bacillus  in  Sepsis. — In  lesions  of  the  intestinal  mucous  mem- 
branes or  in  colon  cystitis,  pyelitis,  or  cholecystitis,  there  is  frequently 
just  before  death  a  terminal  dissemination  of  the  bacilli  and  conse- 
quent septicaemia.  Here  special  symptoms  of  intoxication  may  occur, 
such  as  diarrhoea,  changes  in  temperature,  heart  weakness,  and  hemor- 
rhages. In  most  of  these  cases  infection  proceeds  from  the  in^stines, 
but  in  not  a  few  from  the  wounded  urethra  or  bladder.  The  colon 
septicaemia  is  detected  by  blood  cultures.  At  times  very  few  bacilli 
are  found,  and  then  the  blood  infection  may  be  less  important  than 
the  local  one.  Cases  occurring  in  typhoid  and  cholera  are  often  observed, 
especially  in  relapses  in  typhoid.  In  very  young  infants  a  malignant 
septicaemia  with  tendency  to  hemorrhages  is  due  to  colon  septicaemia. 
An  epidemic  due  to  colon  infection  of  water  has  been  noted.  Infection 
through  food  and  water  are  usually  brought  about  by  other  closely 
allied  bacilli  not  belonging  to  the  colon  group. 

Colon  Bacillus  in  Diarrhoea. — Lesage,  in  1898,  stated  that  25  per  cent, 
of  770  cases  reported  by  him  of  breast-fed  children  were  due  to  pure 
colon  infection,  while  the  others  were  from  mixed  infection  in  which 
thf  meaning  of  the  colon  bacilli  present  was  more  doubtful.  The 
majority  of  bacteriologists  are  inclined  to  doubt  that  the  typical  colon 
bacillus  is  an  important  etiological  factor  in  the  production  of  diarrhoea, 
but  believe  that  it  is  due  rather  to  other,  slightly  different,  micro-organ- 
isms, colon-like  in  their  characteristics.  The  reasons  for  this  opinion 
are  given  by  Escherich  and  Pfaundler  as  follows: 

1.  Animals  are  certainly  affected  by  epidemic  infections  of  bacteria 
closely  allied  to  the  colon  group — e.  g.,  diarrhoea  of  calves  and  cows, 
hog-cholera,  enteritis  with  ulceration  in  horses,  etc. 

2.  The  histories  of  attacks  of  acute  diarrhoea  in  men  after  eating 
food  of  such  infected  animals,  and  the  presence  of  the  serum  reaction 
afterward.      These  bacteria    are  colon-like,  though    classed    with  the 
enteritidis  group. 

3.  The  diseases  of  typhoidal  nature  are  due  to  the  closely  allied 
paracolon  or  paratyphoid  bacilli,  and  others  are  due  to  the  dysentery 
group,  in  which  the  inflammatory  and  necrotic  process  localizes  itself 
mostly  in  the  lower  colon  and  rectum. 


244  BACTERIA  PATHOGENIC  TO  MAN 

Numerous  epidemics  of  acute  diarrhoea  in  children  from  one  to  five 
years  of  age  have  been  noted  in  which  almost  pure  cultures  of  colon 
bacilli  have  been  found.  The  symptoms  begin  with  high  fever  which 
often  rapidly  falls,  and  frequent  stools  containing  mucus  and  streaks 
of  blood  or  only  watery.  These  symptoms  may  quickly  abate  or  go 
on  to  a  toxic  state  characterized  by  heart  weakness  and  drowsiness. 
This  may  lead  to  lung  complications  or  death.  In  many  such  cases  in 
America  when  blood  has  been  present  we  have  found  one  of  the  mannite 
fermenting  types  of  the  dysentery  bacillus.  Here  the  lesions  must  be 
considered  as  being  due  to  mixed  infection. 

The  colon  group  as  exciters  of  inflammation  are  much  less  apt  to 
produce  pus  than  the  pyogenic  cocci.  The  peritoneum,  the  bile  tracts, 
and  the  urinary  tracts  are  most  frequently  affected,  but  it  also  causes 
inflammation  in  wounds  and  various  organs  of  the  body. 

Experimental  evidence  goes  to  show  that  the  injection  of  cultures  of  any 
of  the  varieties  of  colon  bacilli  into  the  peritoneal  cavity  produces  intense 
and  fatal  peritonitis.  Some  varieties  when  freshly  isolated  are  espe- 
cially virulent.  Not  only  perforation  of  the  intestines  in  man,  but  injury 
to  the  intestinal  walls,  allows  colon  infection  of  the  peritoneum  to 
take  place,  and  if  foreign  bodies  are  present  in  the  peritoneum,  or  the 
epithelium  injured,  or  absorption  interfered  with,  such  acute  general 
peritonitis  is  very  probable.  At  first  most  of  these  cases  were  believed 
to  be  a  pure  colon  infection,  but  now  it  is  known  that  this  idea  came 
largely  from  the  overgrowth  of  colon  bacilli  in  the  cultures.  More 
careful  investigations,  through  cultures  and  smears,  have  demonstrated 
the  fact  that  streptococci,  and  less  frequently  staphylococci  and  pneu- 
mococci,  are  present  in  peritonitis  arising  from  intestinal  sources. 
The  colon  bacilli  found  even  in  the  same  case  commonly  comprise  many 
varieties. 

The  Colon  Group  in  Inflammation  of  the  Bile  Tract. — The  contents  of 
the  gall-bladder  are  usually  sterile.  This  is  true  in  spite  of  the  fact 
that  bile  is  apparently  a  good  culture  medium  for  the  colon  group. 
Simple  tying  of  the  neck  of  the  gall-bladder  usually  causes  a  colon 
infection  to  take  place  within  twenty-four  hours.  Obstruction  of  the 
bile-duct  through  various  causes  is  fairly  common  in  man.  The  gall- 
bladder then  becomes  infected,  and  following  the  inflammation  of  the 
mucous  membranes  there  is  often  the  formation  of  gallstones.  Some  cases 
of  jaundice  are  believed  to  be  due  to  colon  inflammation  of  the  gall-ducts. 

Inflammation  of  the  Pancreas. — Welch  was  the  first  to  record  a  case 
-of  pancreatitis  with  multiple  fat  necroses  due  to  colon  infection.  A  few 
more  cases  have  since  been  reported  as  due  to  members  of  the  colon 
group,  either  alone  or  in  conjunction  with  the  pyogenic  cocci. 

Inflammation  of  the  Urinary  Tract. — As  far  back  as  1879  Bouchard 
noted  cystitis  due  to  bacilli  of  the  colon  group.  After  injury  of  the 
bladder  mucous  membrane,  or  by  ligature  of  the  urethra,  it  is  possible 
to  excite  cystitis  in  animals  by  injection  of  colon  bacilli.  When  cystitis 
is  established  the  bacterial  infection  frequently  spreads  to  the  pelvis 
of  the  kidneys,  causing  a  pyelitis  or  suppurative  nephritis.  The  same 


Tin-:  COLON  n.\<  II.LUS  <;ROI  p  245 

process  often  occurs  in  man.  In  most  cases  of  chronic  cystitis  the 
ureters  and  pelves  of  the  kidneys  become  involved;  any  malformation 
of  the  ureters  aids  the  process.  From  the  pelvis  the  bacteria  push  up 
into  the  urinary  tubules  and  excite  inflammation  and  multiple  abscesses. 
At  times  the  bacilli  force  their  way  through  the  lymph  channels  or 
capillaries  into  the  blood  current.  Colon  infection  of  the  different 
parts  of  the  urinary  tract  may  occur  at  any  age,  from  infancy  upward. 
Instead  of  starting  in  the  bladder  it  may  begin  in  the  kidney  itself, 
the  colon  bacilli  coming  from  the  blood  or  peritoneum.  In  many  of 
these  cases  the  bacilli  isolated  from  the  urine  are  clumped  in  high 
dilutions  of  blood  from  the  patient. 

Although  other  bacteria — the  pyogenic  cocci,  the  proteus,  the  typhoid 
bacillus,  etc. — may  excite  cystitis,  still  in  90  per  cent,  of  all  cases  some 
of  the  colon  group  are  found,  and  this  percentage  is  even  higher  in 
young  children.  The  clinical  picture  of  colon  infection  is  very  variable. 
The  lightest  cases  progress  under  the  guise  of  a  bacteriuria.  The  urine 
is  passed  a  little  more  frequently  and  shows  a  fine  granular  cloudiness. 
The  reaction  is  acid.  The  cell  elements  are  but  little  increased.  There 
is  an  excess  of  mucus.  Albumin  is  absent  or  present  in  only  a  trace. 
The  condition  may  last  for  weeks  or  months  and  then  spontaneously 
disappear  or  grow  worse.  With  a  somewhat  more  severe  infection 
there  is  painful  urination,  perhaps  tenesmus,  increase  of  pus  cells, 
and  slight  fever.  In  a  conical  glass  a  sediment  of  pus  cells  forms 
at  the  bottom,  and  clear  urine  remains  above.  In  chronic  cases 
the  fever  is  usually  absent,  but  anaemia  and  loss  of  tone  appear.  If 
the  infection  passes  to  the  kidney  colicky  pain  and  tenderness  over 
the  region  of  the  kidneys  are  usually  present.  The  most  important 
symptom  of  pyelitis  is  an  irregular  intermittent  fever  resembling  malaria. 
The  albumin  is  increased  in  the  urine  and  red  blood  cells  may  be  seen. 
If  a  general  nephritis  arises  the  symptoms  are  all  intensified  and  an 
anaemic  condition  may  develop.  A  septicaemia  may  finally  result. 
Therapeutic  bacteriological  means  are  employed  to  treat  this  affec- 
tion; irrigation  of  the  bladder  with  creolin,  lysol,  and  silver  solution. 
Internally  urotropin  or  salol  are  given. 

In  most  of  these  cases  the  microscopic  examination  is  sufficient 
to  make  a  probable  diagnosis,  since  the  bacteria  are  so  abundant. 
The  variety  of  colon  bacillus  present  can,  of  course,  only  be  told  by 
cultures  and  other  means.  In  the  urine  they  appear  as  diplobacilli, 
or  partly  in  short,  almost  coccus,  forms,  partly  in  long  threads.  As 
a  rule,  motility  is  absent.  Not  infrequently  the  cultures  appear  to  be 
identical  with  the  bacterium  lactis  aerogenes. 

The  characteristics  of  the  urine  itself  have  much  to  do  with  the 
probability  of  infection;  the  more  acid  urines  being  less  likely  to  afford 
a  proper  soil  for  growth.  Some  urines  are  bactericidal  even  when 
they  are  neutral.  The  substances  producing  this  condition  are  not 
knqwn.  The  colon  bacilli  in  the  urine  produce  no  appreciable  effect 
on  the  reaction,  but  give  up  some  of  their  toxins,  which  upon  absorp- 
tion cause  the  deleterious  local  and  general  effects.  The  serum  of  the 


246  BACTERIA  PATHOGENIC  TO  MAN 

patient  usually  agglutinates  the  cultures  from  the  urine  in  1 : 20  or  1 : 50 
dilutions,  but  this  property  is  sometimes  absent,  especially  in  light  cases. 

The  colon  variety  found  in  the  urine  can  ordinarily  be  detected  also 
in  the  feces.  This  would  suggest  an  autoinfection.  Cystic  infection  in 
the  female  usually  takes  place  through  the  urethra.  In  both  male 
and  female  any  injury  or  disease  of  the  rectal  mucous  membranes 
contiguous  to  the  bladder  creates  a  possibility  of  the  invasion  of  the 
colon  through  the  lymph  channel  of  the  inflamed  tissues  and  cause  a 
colon  cystitis.  It  is  also  possible  that  at  times  an  infection  may  take 
place  through  the  kidney,  this  organ  having  received  the  bacilli  from 
the  blood.  Besides  the  above-mentioned  ways,  we  may  have  direct 
infection  carried  in  by  the  catheter,  sound,  etc. 

In  all  cases  in  addition  to  the  introduction  of  the  colon  bacillus  a 
predisposing  condition  must  be  present,  such  as  more  or  less  marked 
retention  of  urine  by  an  enlarged  prostate  or  stricture,  any  unhealthy 
state  of  the  mucous  membrane  or  general  depression  of  vitality. 

The  Colon  Group  as  Pus  Formers. — Members  of  this  group  are  fre- 
quently the  cause  of  abscesses  in  the  region  of  the  rectum,  urethra, 
and  kidney.  They  are  rarely  met  with  in  other  locations. 

Jhe  Colon  Group  in  Inflammation  Not  Previously  Mentioned. — Broncho- 
pneumonia,  lobar  pneumonia,  and  pleurisy  have  occasionally  been 
caused  by  colon  bacilli,  probably  from  blood  sources.  Not  a  few  cases 
of  meningitis  and  spinal  meningitis  in  infants,  following  localized  colon 
infections,  are  due  to  colon  bacilli.  The  symptoms  are  not  well  devel- 
oped, as  a  rule.  Some  cases  of  endocarditis  have  also  been  noted. 

The  Colon  Group  as  Producers  of  Absorbable  Toxins. — Through  intes- 
tinal fermentation  substances  are  formed  which  when  absorbed  produce 
marked  nervous  symptoms.  The  presence  of  indican  in  the  urine 
usually  means  that  such  improper  fermentation  is  present. 

Methods  of  Isolation. — While  the  isolation  of  typhoid  bacilli  from 
feces,  water,  dust,  etc.,  is  attended,  as  a  rule,  with  difficulty,  pure  cul- 
tures of  colon  bacilli  can  usually  be  obtained  from  such  substances  by 
the  simplest  procedures.  The  following  methods  may  be  recommended  : 

1.  Inoculate  10  c.c.  of  fluid  2  per  cent,  lactose  or  dextrose  litmus 
agar  with  feces  or  suspected  material.     The  melted  agar  should  be  at  a 
temperature  of  about  41°  C.    After  shaking  pour  in  Petri  dish.     Several 
dilutions  should  be  made.     After  eighteen  hours  examine  the  plates  and 
inoculate  the  contents  of  a  number  of  tubes  containing  2  per  cent, 
dextrose  agar  with  any  colonies  showing  a  red  color.     The  colon  bacilli 
will  produce  gas  and  acid. 

2.  Inoculation  of  increasing  quantities  of  the  material  (water)  in  2 
per  cent,  dextrose  nutrient  bouillon.     The  presence  of  colon  bacilli  in 
the  inoculated  portion  produces  after  twelve  to  twenty-four  hours  active 
fermentation  in  the  tube.     (Plate  and  isolate  as  in  1.) 

3.  The  inoculation  of  glucose  bouillon  to  which  small  amounts  of 
carbolic  acid  or  of  hydrochloric  acid  have  been  added  to  inhibit  .the 
growth  of  many  other  varieties  of  bacteria.     This  method  has  not  given 
any  better  results  than  2. 


COLOX-TYPHOID  INTERMEDIATES  247 

Intermediate  Members  of  the  Typhoid-colon  Group  of  Bacilli. 

Until  recent  years  the  difficulties  attending  the  differentiation  of  the 
various  members  of  the  typhoid  and  colon  groups  of  bacilli  have  been 
so  great  that  an  accurate  study  of  the  varieties  has  not  been  possible. 
The  application  of  the  Gruber-Widal  reaction  to  this  investigation  has, 
however,  served  to  clear  up  the  field  to  a  great  extent,  so  that  at  the 
present  time  the  differentiation  is  comparatively  easy.  Gartner's  dis- 
covery of  the  bacillus  enteritidis  in  1888  in  association  with  epidemics 
of  meat  poisoning  first  gave  impetus  to  the  study  of  the  intermediates. 
Nocard's  work  on  bacillus  psittacosis  followed  in  1892.  In  1893  Gilbert 
introduced  the  terms  "paracolon"  and  "paratyphoid"  to  designate 
bacilli  of  this  group  resembling  more  nearly  in  biological  characters  the 
colon  bacillus  on  the  one  hand  and  the  typhoid  bacillus  on  the  other, 
but  at  that  time  the  organisms  now  known  as  paratyphoid  bacilli  had 
not  been  identified. 

The  intermediates  include  bacillus  enteritidis  and  similar  organisms 
recovered  from  cases  of  epidemic  meat  poisoning,  the  gas-producing 
typhoid-like  bacilli  of  various  observers,  bacillus  psittacosis,  bacillus 
cholerse  suis,  bacillus  icteroides,  bacillus  of  calf  septicaemia,  and  the 
various  paratyphoid  and  paracolon  bacilli  that  have  been  described 
recently. 

The  bacilli  intermediate  between  bacil!us  coli  communis  and  bacillus 
typhosus  can  be  distinguished  without  difficulty  from  either  of  them. 
They  produce  gas  in  glucose  media  and  in  this  respect  they  differ  from 
typhoid,  but,  unlike  bac  llus  coli  communis,  they  have  no  power  of 
fermenting  lactose,  coagulating  milk  or  forming  indol. 

They  are  not  agglutinated  by  typhoid  sera  except,  as  in  the  case  of 
colon  bacilli,  imperfectly  in  low  dilutions  because  of  group  agglutinin. 

Among  the  intermediates  themselves,  however,  two  main  groups  can 
be  recognized,  and  it  is  proposed  to  call  these  paracolon  and  paratyphoid 
groups,  the  former  appearing  in  some  respects  to  be  more  nearly 
allied  to  bacillus  coli  communis  and  the  latter  more  nearly  to  typhoid. 

The  main  points  of  difference  are  that  the  paracolons  turn  milk  and 
whey  alkaline  after  a  short  initial  acidity  and  form  gas  freely  in  glu- 
cose media,  while  with  the  paratyphoids  there  is  in  milk  and  whey  an 
initial  acidity,  but  no  or  very  slight  subsequent  alkalinity;  the  gas 
production  in  glucose  media  is  much  less  pronounced.  Neutral  red 
agar  also  differentiates  between  the  two  groups.  Like  bacillus  coli  com- 
munis all  the  intermediates  reduce  the  color  to  yellow  in  twenty-four 
to  forty-eight  hours,  but  with  the  paratyphoids  after  four  or  five  days 
the  red  color  begins  to  return  from  above  downward  until  in  two  or 
three  weeks  the  medium  is  again  red  throughout.  With  the  paracolons 
the  yellow  color  is  permanent. 

Agglutination  tests  have  taught  us  that  the  members  of  the  coli  com- 
munis group  do  not  constitute  a  distinct  species  as  in  the  case  with 
typhoid  bacilli.  When  these  tests  are  applied  to  the  intermediates  it 
Is  found  that  the  members  of  the  paracolon  group  do  not  all  show  mutual 


248  BACTERIA  PATHOGENIC  TO  MAN 

reactions,  and  the  group  must,  therefore,  be  composed  of  a  number  of 
distinct  races,  as  is  the  case  with  bacillus  coli  communis.  The  para- 
typhoids, on  the  other  hand,  none  of  which  has  so  far  been  isolated  from 
cases  other  than  typhoidal,  interact  without  exception;  that  is  to  say,  an 
active  serum  prepared  from  any  one  of  the  bacilli  will  agglutinate  all  the 
members  of  the  group.  The  paratyphoids,  then,  appear  to  be  a  distinct 
species  in  the  same  sense  that  the  typhoid  bacilli  are  a  distinct  species. 
It  may  be  found  possible  to  distinguish  various  species  within  the 
paracolon  group.  There  are  certainly  three  members  which  afford 
mutual  reactions — one  of  those  isolated  by  Schottmiiller,  one  by 
Kurth,  and  one  by  Libman — and  these  might  provisionally  be  called 
^-paratyphoids.  Since,  however,  there  are  others  of  the  paracolon 
group  which  have  caused  typhoidal  symptoms,  yet  do  not  appear  to 
belong  to  this  particular  species,  it  seems  a  little  premature  to  attempt 
these  fine  distinctions,  and  for  the  present  it  will  be  sufficient  to  confine 
ourselves  to  the  idea  of  the  two  main  groups : 

I.  The  Paracolons. — A  group  of  bacilli,  the  members  of  which  are 
culturally  alike,  but  constitute  several  distinct  species,  some  of  which 
may  give  rise  to  typhoidal  symptoms  in  man. 

II.  The  Paratyphoids. — A  distinct  species,  culturally  unlike  the  para- 
colon bacilli,  and  causes  typhoidal  symptoms  in  man. 

Pathogenicity  in  man  has  been  established  for  other  of  the  inter- 
mediates. Broadly  speaking  there  are  three  types  of  infection: 

1.  TYPHOID  TYPE,  caused  by  the  paratyphoid  bacilli  and  certain  of 
the  paracolons.1 

2.  EPIDEMIC  MEAT-POISONING  TYPE,  caused   by  bacillus  enteritidis 
and  its  allies. 

3.  PSITTACOSIS  TYPE,,  caused  by  bacillus  psittacosis. 

In  addition  to  these  three  types  Griinbaum  has  suggested  that  febrile 
jaundice  may  be  caused  by  one  of  the  intermediates. 

Bacillus  icteroides  (Sanarelli),  which  is  probably  an  intermediate,  is 
no  longer  the  accredited  cause  of  yellow  fever,  but  is  considered  simply 
as  a  secondary  invader.  It  is  stated  that  it  ferments  dextrose  and 
saccharose. 

THE  TYPHOID  TYPE.  The  Term  Paratyphoid  Fever. — Though 
Gilbert  introduced  the  terms  "paracolon"  and  "paratyphoid"  in 
1893  to  designate  groups  of  bacilli,  Achard  and  Bensaude  (1896)  were 
the  first  to  employ  the  term  paratyphoid  in  a  clinical  sense.  This  use 
of  the  term  was  sanctioned  by  Schottmiiller  in  1901,  and  has  been 
adopted  by  several  recent  writers.  On  the  other  hand,  Coleman  seriously 
questions  whether  the  term  should  be  recommended,  as  it  leads  to 
unfortunate  multiplicity.  He  considers  it  proven  that  infection  with 
paratyphoid  bacilli  is  often  manifested  by  symptoms  practically  identical 
with  typhoid  fever  except  for  the  Widal  reaction,  that  there  are  differ- 
ences (biological  and  in  serum  reactions)  even  among  the  paratyphoid 
bacilli  themselves  (/9-paratyphoids),  and  that  bacilli  of  the  enteritidis 

1  Petruschky's  bacillus  fsecalis  alcaligenus,  while  not  an  intermediate  of  the  group  (Durham),  may 
also  produce  typhoidal  symptoms. 


COLON-TYPHOID  INTERMEDIATES  249 

type  may  at  times  produce  typhoidal  symptoms.  He  believes  it  is  no 
more  advisable  to  make  a  clinical  subdivision  of  these  cases  than  of  the 
cases  of  pneumonia  or  infective  endocarditis  which  may  be  due  to  one 
of  several  different  micro-organisms.  Paratyphoid  infections  do  not 
constitute  a  clinical  entity.  There  is  at  least  as  great  diversity  among 
the  different  types  of  typhoid  fever  as  between  typhoid  fever  and  para- 
typhoid infections.  Moreover,  typhoidal  symptoms  may  be  produced 
by  Petruschky's  bacillus  faecalis  alcaligenus  (the  author  states  that  it 
was  obtained  from  the  feces  of  patients  suspected  to  have  typhoid  fever) 
and  yet  this  bacillus  is  not  an  intermediate.  It  lies  just  without  the 
group  on  the  typhoid  side,  in  that  it  does  not  acidify  any  sugar-contain- 
ing medium  (Dunham).  It  is  true  that  the  intestinal  lesions  are  different 
in  that  Peyer's  patches  are  not  usually  markedly  involved.  Even  when 
fatal  hemorrhages  occur  there  are  usually  found  only  one  or  more  deep 
erosions.  The  average  course  is  milder  and  the  method  of  infection 
somewhat  different. 

These  various  considerations  make  it  necessary  to  abandon  the  idea 
of  the  specificity  of  the  clinical  disease  typhoid  fever.  As  in  the  case 
of  abscesses  the  physician  recognizes  the  clinical  fact,  the  bacteriologist 
determines  the  causative  agent.  It  certainly  seems  better  to  confine  the 
terms  "paratyphoid"  and  "paracolon"  to  the  domain  of  bacteriology 
and  to  hospital  practice,  where  bacteriological  examinations  can  be 
carefully  made,  and  to  broaden  the  scope  of  the  etiology  of  typhoid 
fever  to  include  these  several  organisms — bacillus  fsecalis  alcaligenus  (?), 
bacillus  typhosus,  bacillus  paratyphosus,  and  certain  members  of  the 
paracolon  group  (^-paratyphoids). 

Geographical  Distribution  and  Relative  Frequency  of  Paratyphoid 
Infection. — The  cases  have  been  widely  distributed  geographically, 
having  occurred  in  Paris,  Hamburg,  Bremen,  Strassburg,  Liverpool, 
Philippine  Islands,  New  York  City,  Baltimore,  and  Philadelphia. 

Very  little  can  be  said  of  the  relative  frequency  of  paratyphoid  infec- 
tions. Gwyn's  case  was  the  only  one  of  265  cases  which  failed  to  give 
the  Widal  reaction.  Six  of  Schottmuller's  cases  occurred  in  a  series 
of  68  and  Kurth's  5  in  a  series  of  62  cases  whose  sera  were  tested  for 
the  Widal  reaction.  Johnston's  4  cases  were  found  among  194,  and 
Hewlett's  1  in  a  series  of  26  cases  of  typhoid  fever.  Hiinermann  has 
reported  an  epidemic  of  38  cases  of  paratyphoid  infection  occurring  in 
the  garrison  at  Saarbriick.  Falcioni  reports  5  cases  out  of  100  cases 
of  supposed  typhoid  fever.  The  proportion  of  negative  Widal  reactions 
is  low  in  the  "statistics,  but  there  is  a  source  of  error  here  in  that  until 
very  recently  the  tests  have  not  been  made  in  high-enough  dilutions — 
that  is,  at  least  as  high  as  1 : 40. 

Post-mortem  Findings. — Autopsies  were  performed  on  3  fatal  cases 
(Strong,  Longcope,  Tuttle).  The  interest  in  these  autopsies  naturally 
centres  on  the  condition  of  the  intestine.  Strong  states  that  both  the 
large  and  small  intestines  were  normal  throughout  except  for  moderate 
catarrh  and  a  few  superficial  hemorrhages.  The  solitary  and  agmin- 
ated  follicles  showed  no  lesions.  The  mesenteric  lymphatics,  how- 


250  BACTERIA  PATHOGENIC  TO  MAN 

ever,  and  some  along  the  small  intestine,  were  hemorrhagic.  In  Long- 
cope's  case  the  intestine  showed  no  changes  either  on  gross  or  micro- 
scopic examination.  The  spleen  in  both  cases  was  enlarged.  The 
other  pathological  changes  were  those  common  to  febrile  conditions.  In 
Tuttle's  case  a  few  erosions  just  above  the  ileocsecal  valve  were  present. 

SOURCE  OF  INFECTING  BACILLI. — Tuttle's  case  happened  to  be  a 
laboratory  employe  in  the  service  of  the  Department  of  Health  and 
was  carefully  investigated  by  us.  We  found  that  two  families  consisting 
of  eleven  members  drank  water  from  an  open  uncovered  tank.  During 
the  summer  the  tank  was  not  cleaned  and  was  only  occasionally  filled 
by  pumping  in  water  from  the  city  supply.  Sometimes  the  water  was 
the  color  of  tea.  During  a  single  week  four  members  of  one  family 
and  three  of  the  other  were  stricken  with  a  typhoid-like  fever.  The 
two  families  had  no  social  intercourse  with  each  other. 

Symptomatology. — It  is  a  significant  fact  that  many  of  the  reported 
cases  of  paratyphoid  infection  were  considered  to  be  genuine  typhoid 
fever  without  the  Gruber-Widal  reaction  until  a  bacteriological  study 
revealed  their  true  nature. 

Intestinal  hemorrhages,  furunculosis,  initial  bronchitis,  cystitis, 
pyelonephritis  (?),  purulent  arthritis,  bronchopneumonia,  and  venous 
thrombosis  have  been  reported  as  complications.  Osteomyelitis  is  the 
only  recorded  sequel. 

The  duration  of  the  disease  has  varied  from  twelve  to  eighty-four 
days,  with  a  majority  of  the  cases  continuing  between  twenty  and 
thirty-six  days.  Some  of  the  cases  have  been  of  short  duration,  lasting 
from  twelve  to  eighteen  days. 

The  Serum  Reaction  in  Cases  of  Paratyphoid  Infection. — Since  the 
introduction  of  serum  reactions  as  a  means  of  diagnosis,  it  has  been 
a  well-recognized  fact  that  a  small  proportion  of  cases  which  are  clinic- 
ally typhoid  fever  fail  to  give  the  reaction.  Brill,  adding  to  Cabot's 
statistics,  finds  that  of  4879  cases  4781,  or  97.9  per  cent.,  gave  the  reac- 
tion. Gwyn  gives  99.6  per  cent,  as  the  percentage  of  positive  reactions 
in  the  Johns  Hopkins  Hospital.  On  the  contrary,  in  most  of  the  reported 
cases  of  paratyphoid  infection  a  reaction,  except  with  low  dilutions, 
against  the  bacillus  typhosus  has  been  absent.  It  is  probable,  then, 
that  some  at  least  of  the  typhoid  cases  with  negative  reaction  were 
really  paratyphoid  infection. 

On  the  other  hand,  it  cannot  be  assumed  that  all  cases  clinically 
typhoid  fever,  which  have  been  reported  as  giving  the  Gruber-Widal 
reaction,  were  cases  of  genuine  typhoid  infection.  The  brilliant  work 
of  Dunham  on  the  typhoid-colon  group  of  bacilli  and  its  serum  reac- 
tions has  brought  out  the  fact  that  certain  members  of  this  group  may 
be  mutually  interacted  upon  by  sera  of  infected  patients  and  of 
immunized  animals.  This  is  especially  true  of  sera  in  low  dilution. 
Since  in  the  earlier  years  of  the  Gruber-WTidal  reaction  the  technique 
had  not  been  worked  out,  and  dilutions  were  more  frequently  low  than 
not,  some  of  the  cases  reported  as  typhoid  fever  may  have  been  infec- 
tions with  paratyphoid  bacilli. 


COLOX-TYPHOID  INTERMEDIATES  251 

Diagnosis. — The  only  reliable  criteria  for  diagnosis  are  absence  of 
the  Gruber-Widal  reaction  in  proper  dilution  (not  less  than  1 : 40)  with 
a  positive  reaction  against  a  known  paratyphoid  bacillus  or  the 
recovery  of  a  paratyphoid  bacillus  from  the  blood,  urine,  or  compli- 
cating inflammatory  process. 

The  clinical  type  of  the  disease  is  of  little  value  in  a  single  case.  It 
has  already  been  stated  that  the  reported  cases  of  paratyphoid  infection 
have  been  both  mild  and  severe. 

The  cases  of  paratyphoid  infection  are  too  few  to  state  what  the 
prognosis  should  be.  It  can  only  be  said  that  the  majority  of  the  cases 
have  been  mild,  though  there  have  been  about  9  per  cent,  of  deaths 
among  the  cases  reported. 

EPIDEMIC  MEAT-POISONING  TYPE. — Gartner  announced  his  dis- 
covery of  bacillus  enteritidis  as  the  cause  of  epidemic  meat  poisoning 
in  1888.  A  cow  sick  for  two  days  with  profuse  diarrhoea  had  been 
slaughtered  in  Saxony  and  the  meat  sold  for  food.  Of  the  persons  who 
ate  of  the  meat  57  became  ill,  and  1  died.  Gartner  recovered  the 
bacillus  from  the  meat  and  from  the  organs  in  the  fatal  case. 

Previous  to  Gartner's  discovery  the  cause  of  meat  poisoning  had 
been  held  to  be  bacterial  products,  and  while  this  may  still  be  true  in 
certain  instances  there  is  no  satisfactory  evidence  to  support  the  con- 
tention. All  cases  in  which  a  thorough  bacteriological  examination  has 
not  been  made  must  be  excluded. 

Two  kinds  of  bacilli  are  concerned  in  the  production  of  meat  poison- 
ing: 1.  Anaerobic  bacillus  botulinus  of  Van  Erminghem,  a  saprophyte. 
2.  Bacillus  enteritidis  of  Gartner,  including  the  different  strains  of 
this  organism. 

Of  these  bacilli,  bacillus  enteritidis  is  the  more  important,  having 
been  concerned  in  the  greater  number  of  epidemics,  and  causing  true 
meat  poisoning.  It  seems  advisable,  however,  to  say  a  few  words,  by 
way  of  distinction,  on  infection  by  bacillus  botulinus. 

"Botulism,"  "allantiasis,"  and  "sausage  poisoning"  are  the  names 
given  to  infection  by  bacillus  botulinus.  The  infection  of  the  meat 
takes  place  after  the  animal  has  been  slaughtered.  The  meat  is  of 
unsound  appearance  and  odor  and  can  readily  be  seen  to  be  unfit 
for  food. 

The  symptoms  begin  from  twelve  to  twenty-four  hours  after  inges- 
tion  of  the  meat,  with  repeated  attacks  of  vomiting  and  abdominal 
pain.  Soon  the  characteristic  symptoms  appear:  partial  or  complete 
paralysis  of  the  inner  and  outer  recti  muscles  of  the  eye,  and  disturb- 
ances of  the  innervation  of  the  pharynx  and  larynx.  These  are  mani- 
fested by  imperfect  vision,  difficulty  of  speech  and  deglutition,  and 
dry  ness  of  the  throat.  There  are"  no  disturbances  of  sensation  or 
impairment  of  consciousness  and  the  disease  runs  its  course  without 
fever.  Constipation  and  retention  of  urine  follow;  dyspnoea  and  car- 
diac failure  appear,  and  bulbar  paralysis  may  supervene,  causing  death. 
In  earlier  years  the  mortality  from  sausage  poisoning  was  from  30  per 
cent,  to  50*  per  cent.,  but  this  has  been  much  reduced  through  a  better 


252  BACTERIA  PATHOGENIC  TO  MAN 

understanding  of  the  disease.  It  may  be  prevented  by  thoroughly 
cooking  the  meat  and  by  refusing  to  accept  from  the  butcher  meat 
that  is  the  least  tainted. 

True  Meat  Poisoning. — This  form  of  meat  poisoning  is  due  to  bacillus 
enteritidis,  and  in  every  instance  the  animal  is  diseased  at  the  time  of 
the  slaughter.  It  may  be  contracted  by  eating  sausage,  since  the  meat 
of  diseased  animals  is  sometimes  surreptitiously  put  on  the  market 
in  the  form  of  sausage. 

Dunham  makes  bacillus  enteritidis  the  chief  type  of  the  intermediates 
and  proposes  the  name  "the  enteritidis  group."  Buxton  classes  the 
bacillus  with  the  paracolons.  It  does  not  ferment  lactose ;  milk  becomes 
more  alkaline;  it  ferments  dextrose  with  a  production  of  gas  containing 
about  one-third  CO2,  two-thirds  H,  and  it  also  ferments  mannite, 
maltose,  and  dextrin. 

Bacillus  enteritidis  is  pathogenic  for  cows,  horses,  pigs,  goats,  mice,, 
and  guinea-pigs,  but  not  for  dogs  and  cats. 

The  Injected  Meat. — In  many  epidemics  bacillus  enteritidis  has  been 
isolated,  not  only  from  the  organs  of  fatal  cases,  but  from  the  suspected 
meat.  The  meat  does  not  differ  in  appearance  or  taste  from  that  of 
healthy  animals.  It  has  already  been  stated  that  it  may  be  made  into 
sausages,  and  one  epidemic  at  least  has  been  caused  by  eating  "dried 
meat"  consisting  of  large  pieces  of  the  flesh  of  sheep  and  goats  nearly 
dried  in  the  sun  and  eaten  cooked  or  merely  softened  by  soaking. 
Cooking  does  not  always  destroy  the  bacilli,  as  the  thermal  death 
point  may  not  be  reached  in  the  interior  of  the  meat.  Infected  meat 
which  is  not  eaten  immediately  after  it  has  been  cooked  is  especially 
dangerous. 

The  meat  has  always  come  from  animals  sick  at  the  time  of  slaugh- 
ter. The  meat  of  cows  and  calves  have  most  often  caused  the  epi- 
demics, though  that  of  horses,  pigs,  and  goats  have  also  been  respon- 
sible. Dunham  says  that  no  known  case  has  come  from  mutton,  and 
that  the  pig  has  .been  implicated  in  only  one  outbreak  which  has  been 
studied  bacteriologically.  In  this  connection  it  is  interesting  to  recall 
that  Theobald  Smith  has  insisted  on  the  similarity  between  the  hog- 
cholera  bacillus  and  bacillus  enteritidis. 

The  animals  from  which  the  infected  meat  has  come  have  suffered 
during  life  from  puerperal  fever  aixd  uterine  inflammations,  navel 
infection  in  calves,  septicaemia,  septicopysemia,  diarrhoea,  and  local 
suppurations,  and  have  not  infrequently  been  killed  because  of  their 
unsound  condition.  How  animals  become  infected  is  not  known. 

Dunham  thinks  milk  may  be  a  source  of  infection  in  man,  but 
states  that  bacteriological  evidence  of  it  is  incomplete.  Bacillus 
enteritidis  has  been  found,  however,  in  the  milk  of  infected  guinea-pigs 
(Basenau). 

Transmission  to  Man. — The  disease  may  be  transmitted  to*  man  in 
two  ways:  (1)  by  eating  the  infected  meat,  and  this  is  by  far  the  most 
common  means,  and  (2)  from  man  to  man  according  to  Gartner,  Van 
Erminghem,  and  Fischer.  Dunham  found  inconclusive  evidence  of  this- 


Till:  D)  SI-XTERY  BACILLI  253 

means  of  transmission  in  one  epidemic.  Fischer  thinks  transmission 
may  take  place  through  the  excreta.  It  will  subsequently  be  seen  that 
psittacosis  may  be  transmitted  from  man  to  man. 

Epidemics  of  meat  poisoning  may  occur  in  any  season,  but  are  more 
frequent  during  the  warm  months. 

Symptomatology. — While  the  characteristic  symptoms  of  sausage 
poisoning  relate  to  the  nervous  system,  in  true  meat  poisoning  they 
an-  gastrointestinal.  Fischer  divides  meat  poisoning  into  three  clinical 
forms:  (1)  typhoidal;  (2)  choleraic;  (3)  gastroenteric. 

Prevention. — Since  neither  appearance  nor  taste  affords  any  clue 
to  the  noxious  quality  of  tiie  infected  meat,  its  unfitness  for  food  can 
only  be  told  through  bacteriological  examination  or  a  knowledge  of  its 
source.  Dunham  states  that  thorough  cooking  will  kill  the  bacilli, 
but  it  must  be  remembered  that  in  this  process  the  thermal  death  point 
of  the  bacilli  may  not  be  reached  in  the  innermost  portions  of  the  meat. 

Bacillus  Faecalis  Alcaligenus. 

This  bacillus  resembles  a  colon  bacilus  which  has  lost  its  power  to 
ferment  sugars.  Morphologically  it  resembles  the  typhoid  bacillus.  It  is 
frequently  present  in  the  intestines  and  may  have  pathogenic  properties. 


The  Dysentery  Bacillus — The  Paradysentery  Bacilli  (Mannite 
Fermenting  Types). 

Dysentery  may  be  divided  into  acute  and  chronic.  Amoebae  appear 
to  be  the  chief  exciting  factor  in  most  cases  of  chronic  dysentery,  though 
bacilli  of  the  colon  group  also  play  a  part. 

In  temperate  climates  acute  dysentery  is  but  very  rarely  due  to 
amcjebse,  but  usually  to  the  bacilli  identified  by  Shiga  or  to  allied 
bacilli  identified  by  Kruse,  Flexner,  and  Park.  By  dysentery  we  mean 
a  definite  symptom  complex;  it  is  not  an  etiological  term.  In  acute 
dysentery  the  onset  is  sudden  and  ushered  in  by  cramps,  diarrhea, 
and  tenesmus.  The  stools  at  first  feculent,  then  seromucous,  become 
bloody  or  composed  of  coffee-ground  sediment.  At  the  height  of  the 
disease  there  are  ten  to  fifty  stools  in  the  twenty-four  hours.  After 
two  to  seven  days  the  blood  usually  disappears.  In  temperate  climates 
the  mortality  varies  from  5  to  20  per  cent. 

In  severe  cases  in  adults  the  lesions  are  of  a  diphtheritic  character 
and  may  be  very  marked.  In  young  children,  even  in  fatal  cases,  the 
lesions  may  be  more  superficial.  The  following  macroscopic  and 
microscopic  report  of  the  intestinal  findings  on  a  fatal  case  of  bacillary 
dysentery  in  an  infant  is  a  typical  picture: 

Small  Intestines. — Slightly  distended.  Mesenteric  glands  large  and 
red. 

Large  Intestines. — Outer  surface  vessels  congested  and  prominent. 
On  section  covered  with  a  yellowish  mucus.  Mucous  membrane  seems 


254  BACTERIA  PATHOGENIC  TO  MAN 

to  be  absent  in  places.  Solitary  follicles  are  elevated  and  enlarged> 
especially  in  the  region  of  sigmoid  flexure.  In  some  instances  the 
centre  of  the  follicles  are  depressed  and  appear  to  be  necrotic. 

Appendix. — On  section  lymphatic  follicles  are  swollen  with  depressed 
centres  similar  to  the  condition  described  in  the  large  intestines. 

Small  Intestines. — Peyer's  patches  are  distinctly  swollen,  but  in  no 
instance  is  there  ulceration  or  necrosis. 

Large  Intestine. — Mucous  glands  are  for  the  most  part  normal,  but 
over  the  solitary  follicles  they  have  broken  down  somewhat  and  con- 
tain polynuclear  leukocytes.  The  interglandular  stroma  in  these  places 
has  undergone  necrosis.  The  necrotic  area  extends  down  into  the 
submucosa  in  the  region  of  the  solitary  follicles.  The  capillaries  of 
the  solitary  follicles  are  much  dilated  and  congested.  The  submucosa 
is  thickened  and  slightly  cedematous.  The  connective-tissue  cells  appear 
to  have  undergone  a  slight  hyaline  degeneration.  .The  musculature  is 
not  affected,  neither  is  the  peritoneal  coat. 

Small  Intestines. — Normal. 

Historical  Notes. — In  1897  Shiga  found  in  the  stools  of  cases  of 
dysentery  a  bacillus  which  had  not  been  before  identified.  This 
bacillus  had  many  of  the  characteristics  of  the  colon  bacillus,  but  dif- 
fered from  it,  lacking  motility  and  failing  to  produce  gas  from  the 
fermentation  of  sugar.  It  also  was  entirely  distinct  in  its  agglutination 
characteristics  and  in  its  pathogenic  properties.  Shiga  found  this 
bacillus  present  in  all  the  cases  of  epidemic  dysentery  that  he  exam- 
ined. It  was  most  abundant  during  the  height  of  the  disease  and  dis- 
appeared with  the  return  of  fecal  stools.  It  was  not  found  in  the  stools 
of  healthy  persons.  He  found  that  the  blood  of  dysenteric  patients 
contained  substances  which  agglutinated  the  bacilli  that  he  had 
isolated.  The  serum  from  healthy  individuals  did  not  agglutinate  the 
bacilli  to  any  such  degree  as  the  serum  from  those  sick  with  dysentery. 
When  the  mucous  membrane  of  the  colon  was  examined  in  fatal  cases 
dying  in  the  height  of  the  disease,  the  bacilli  were  found  in  the  super- 
ficial layers  in  almost  pure  cultures.  In  his  hands  a  serum  produced 
by  immunizing  horses  through  injections  of  dysentery  bacilli  gave 
beneficial  results  when  used  in  the  treatment  of  those  ill  with  the 
disease.  A  criminal  fed  with  a  culture  of  the  bacillus  developed 
typical  dysentery.  Certain  animals,  such  as  dogs,  when  subjected  to 
treatment  which  made  them  more  susceptible,  were  attacked  with 
dysentery  after  feeding  on  cultures.  This  was  fairly  similar  to  that 
in  man. 

Morphological  and  Cultural  Characteristics  of  Dysentery  Bacilli.  MICRO- 
SCOPIC.— Similar  to  bacilli  of  the  colon  group. 

STAINING. — Similar  to  bacilli  of  the  colon  group. 

MOTILITY. — No  definite  motility  has  been  observed.  The  molecular 
movement  is  very  active. 

FLAGELLA. — True  flagella  have  not  been  observed  by  most  examiners. 
On  a  very  few  bacilli  in  suitable  smears  Goodwin  demonstrated  terminal 
flagella.  Spores  are  not  formed. 


THE  DYSENTERY  BACILLI 


255 


APPEARANCE  OF  CULTURES.^OH  gelatin  the  colonies  appear  more 
like  the  typhoid  than  the  colon  bacilli.  Gelatin  is  not  liquefied.  On 
agar  growth  is  somewhat  more  delicate  than  the  average  colon  cultures. 

On  Potato.—  A  delicate  growth  just  visible  or  distinctly  brownish. 

In  Bouillon—  Diffuse  cloudiness  with  slight  deposit  and  sometimes 
a  pellicle.  Indol  not  produced  or  in  a  trace  only. 

In  glucose  bouillon  no  production  of  acid  or  gas. 

Neutral  red  agar  is  not  blanched. 

In  Liimus  Milk.  —  After  twenty-four  to  forty-eight  hours  this  becomes 
a  pale  lilac.  Later,  three  to  eight  days,  there  is  a  return  to  the  original 
pale  blue  color.  The  milk  is  not  otherwise  altered  in  appearance. 

Animal  Tests.  —  No  characteristic  lesions  have  followed  swallowing 
large  quantities  of  bacilli.  Dogs  at  times  have  had  diarrhoea  with  slimy 
stools,  but  section  showed  merely  a  hypersemia  of  the  small  intestine. 

FIG.  86  FIG.  87 


"  *•*     ';"« 

•  /•     ,  •  .  4'  .." 

*  ' 


"*  *'      ' 

%  -    -V 

:.-:  ^  /    ; 
•  I 

Dysentery  bacillus.  Colony  of  dysentery  bacilli  in  gelatin. 

Many  animals  are  very  sensitive  to  bacilli  injected  in  vein  or  peri- 
toneum; 0.1  mg.  of  agar  culture  injected  intravenously  produced 
diarrhoea,  paralysis,  and  death;  0.2  mg.  under  the  skin  have  killed,  and 
the  same  amount  in  the  peritoneum  has  caused  bloody  peritonitis,  with 
lowered  temperature  and  diarrhoea.  Both  small  and  large  animals  are 
very  sensitive  to  killed  cultures. 

The  autopsy  of  animals  dying  quickly  from  injection  into  the  peri- 
toneum of  living  or  dead  bacilli  shows  the  peritoneum  to  be  hyper- 
aemic,  the  cavity  more  or  less  filled  with  serous  or  bloody  serous  exudate. 
The  liver  is  frequently  covered  with  fibrinous  masses.  The  spleen 
is  moderately  or  not  at  all  swollen.  The  small  intestine  is  filled  with 
fluid,  the  large  intestine  is  usually  empty.  The  mucous  membrane  of 
both  is  hypersemic  and  sometimes  contains  hemorrhages.  Conradi 
found  ulcer  formation  in  one  case. 

Subcutaneous  injections  of  dead  or  living  cultures  are  followed  by 
infiltration  of  tissues  and  frequently  by  abscess  formation.  The 
dysentery  bacilli  produce  both  extracellular  and  cellular  toxins,  the 
latter  being  the  most  abundant.  The  elimination  of  these  toxins  from 


256  BACTERIA  PATHOGENIC  TO  MAX 

the  body  is  supposed  to  take  place  through  the  intestines,  and  this 
gives  rise  to  the  intestinal  lesions  in  animals  injected  intravenously  or 
intraperitoneally.  The  dysentery  bacilli  are  not  found  in  the  blood 
or  organs  of  animals. 

Paradysentery  Bacilli  as  Exciters  of  Dysentery. — In  1900  Flexner  and 
Strong,  when  in  the  Philippine  Islands,  isolated  bacilli  from  dysen- 
teric stools  which  were  identical  with  the  Shiga  cultures.  At  first  all 
the  cultures  were  supposed  to  be  of  the  Shiga  type,  but  later  among 
those  isolated  bacilli  were  found,  which  differed  from  Shiga's  in 
many  characteristics.  In  the  same  year  Kruse,  in  Germany,  obtained 
from  dysenteric  cases  in  an  asylum  bacilli  which  appeared  to  him  to 
be  culturally  like  those  isolated  by  Shiga,  but  to  differ  in  their  agglu- 
tinating characteristics.  These,  like  those  isolated  by  Flexner,  were 
later  found  to  differ  in  many  characteristics.  In  1902  Duval  and  Bas- 
sett,  in  Baltimore,  thought  they  had  found  the  Shiga  bacilli  in  the  stools 
of  a  number  of  cases  of  summer  diarrhoea.  These  later  proved  to  be 
identical  with  some  of  the  bacilli  isolated  by  Flexner  in  Manila. 
During  the  same  summer  Park  and  Dunham  isolated  a  bacillus  from  a 
severe  case  of  dysentery  occurring  during  an  epidemic  at  Seal  Harbor, 
Mt.  Desert,  Maine,  which  they  showed  to  differ  from  the  Shiga  bacillus 
in  that  it  produced  indol  in  peptone  solution  and  differed  in  agglutin- 
ating characteristics.1  They  at  first  considered  it  identical  with  the 
Philippine  culture  given  them  by  Flexner,  but  in  January,  1903,  it 
was  shown  by  Park  to  be  a  distinct  variety,  and  later  found  by  him 
to  be  the  exciting  factor  in  a  large  number  of  cases. 

Martini  and  Lentz2  published  the  results  of  their  work  in'  Decem- 
ber, 1902.  They  showed  that  the  Shiga  type  of  bacilli  obtained  from 
several  separate  epidemics  in  Europe  agreed  with  the  original  Shiga 
culture  in  that  it  did  not  ferment  mannite.  The  cultures  of  this 
type  agreed  with  each  other  in  agglutinating  characteristics.  When 
the  bacilli  from  Flexner,  Strong,  Kruse,  Park,  Duval  and  others, 
which  differed  from  the  Shiga  culture  in  their  agglutinins,  were  tested 
they  were  all  found  to  ferment  mannite.  Martini  and  Lentz  considered 
that  the  Shiga  bacillus  was  the  true  dysentery  type  and  that  the  man- 
nite fermenting  variety  or  varieties  might  be  mere  saprophytes,  or 
perhaps  be  a  factor  in  the  less  characteristic  cases. 

In  January,  1903,  Hiss3  and  Russell,  independently  of  others,  showed 
that  a  bacillus  isolated  by  them  from  a  dysenteric  stool  differed  from 
Shiga's  bacillus  in  the  same  characteristics  as  mentioned  by  Martini 
and  Lentz. 

At  the  beginning  of  the  summer  of  1903,  therefore,  it  was  established, 
although  not  fully  recognized,  that  there  were  in  dysenteric  stools  at 
least  two  distinct  types  of  bacilli,  the  true  Shiga  type  and  the  type  fer- 
menting mannite  and  producing  indol.  It  had  also  been  established 
that  the  second  type  contained  more  than  one  variety. 

•  New  York  University  Bulletin  of  the  Medical  Sciences,  October,  1902,  p.  187. 
2  Zeitschrift  f.  Hygiene  u.  Infectioriskrank.,  1902,  xli.,  540  and  559. 
:)  Medical  News,  1903,  Ixxxii.,  289. 


THE  DYSENTERY  BACILLI  257 

The  German  observers  considered  the  Shiga  type  as  the  only  one 
which  had  established. its  causal  relation  to  acute  dysentery,  while  the 
American  observers  generally  considered  both  types  to  have  equal 
standing  and  some1  of  them  considered  these  differences  as  not  impor- 
tant and  perhaps  not  permanent.  This  latter  opinion  seems  to  have 
been  held  by  Shiga.2 

We  took  up  the  investigation  at  this  point  with  the  object  of  care- 
fully studying  the  bacilli  isolated  by  us  from  acute  dysentery,  which 
occurred  in  a  number  of  widely  separated  epidemics.  We  hoped  thus 
to  determine  whether  the  bacilli  exciting  acute  dysentery  in  the  Eastern 
States  belonged  to  a  few  distinct  types  or  were  divided  into  a  large 
number  of  varieties. 

In  the  most  extensive  epidemic  that  has  recently  occurred  in 
the  region  of  New  York  City  there  were  in  all  some  500  cases 
of  acute  typical  dysentery.  Whole  families  were  attacked  with  the 
disease. 

The  majority  of  the  cases  were  of  moderate  severity,  the  dysenteric 
discharges  lasting  from  one  to  two  weeks.  There  were  a  number  of 
light  cases,  but  all  had  dysenteric  stools  containing  mucus  and  blood. 
The  mortality  was  about  6  per  cent.  Judging  from  the  cases  investi- 
gated by  us,  over  one-half  of  those  attacked  seem  to  have  been  infected 
by  the  Shiga  type,  and  these  were,  as  a  rule,  the  most  severe  cases. 
Most  of  the  cases  in  two  severe,  though  localized,  epidemics  in  a  Penn- 
sylvania town  and  at  Sheepshead  Bay  were  also  due  to  this  type.  The 
mortality  was  higher  in  these  epidemics.  The  facts  published  abroad 
also  indicate  that  this  variety  has  been  found  in  the  chief  epidemics  in 
Europe  and  Asia.  The  bacilli  isolated  in  the  severe  epidemic  of  dysentery 
reported  by  Vedder  and  Duval  (at  New  Haven,  Conn.)  were  chiefly  of 
this  type.  We  have  never  yet  succeeded  in  isolating  bacilli  which  had 
all  the  characteristics  of  the  Shiga  variety  from  any  diarrhoea  cases  in 
which  no  dysenteric  symptoms  appeared. 

We  turn  now  to  the  mannite  fermenting  varieties,  whose  relationship 
to  dysentery  is  still  doubted  by  some. 

The  cultures  isolated  by  us  from  over  40  cases  were  found  to  fall 
largely  into  two  distinct  types,  one  of  which  differs  from  the  Shiga 
bacillus  more  radically  than  the  other. 

The  variety  nearer  to  the  Shiga  bacillus  has  the  characteristics  of 
the  culture,  which  was  isolated  by  us  at  Seal  Harbor,  Maine,  in 
August,  1902.  The  other  variety  is  represented  by  the  Flexner 
Philippine  type. 

The  first* type  differs  from  the  Shiga  bacillus  in  its  agglutinating 
characteristics  and  in  that  it  produces  considerable  indol  in  peptone 
solution  and  ferments  mannite  with  the  production  of  acids.  The 
second  type  differs  in  these  points  and  in  addition  that  in  its  aggluti- 
nating characteristics  ferments  saccharose  and  chemically  pure  maltose 
in  peptone  solution. 

i  University  of  Pennsylvania  Medical  Bulletin,  July  and  August,  1983. 
*  Zeitschrift  f.  Hygiene  u.  Infectionskrank.,  1902,  xli.,  356. 
17 


258  BACTERIA  PATHOGENIC  TO  MAN 

Besides  the  epidemic  at  Seal  Harbor,  numerous  cases  of  moderately 
severe  or  slight  dysentery  due  to  the  first  type  were  met  with  in  the 
extensive  epidemic  which  has  been  already  alluded  to  in  the  towns 
north  of  New  York  City.  A  few  characteristics  in  many  slightly 
developed  cases  of  dysentery  in  New  York  City  during  the  past  two 
summers  were  caused  by  this  type  of  bacillus.  A  great  many  cases 
are  also  due  to  the  Philippine  type.  A  number  of  rather  severe  cases 
of  dysentery  developed  in  Orange,  N.  J.  Cultures  from  two  cases  were 
made,  and  this  type  alone  obtained.  The  folloAving  is  a  typical  case. 
Eighteen  out  of  thirty  colonies,  selected  from  the  plates,  when  tested 
proved  to  be  dysentery  bacilli  of  the  Philippine  type. 

Dorothy  D.,  aged  two  years  and  three  months.  Seen  first  July  29th, 
a  day  after  the  child  had  eaten  green  apples.  Previous  to  this  the 
child  had  had  an  attack  of  vomiting  and  diarrhoea,  the  sickness  lasting 
two  weeks;  the  diarrhoea  had  subsided  only  two  days  before  present 
illness.  No  blood  was  seen  during  this  first  attack.  When  first  seen, 
the  child  had  a  temperature  of  104.6°,  with  vomiting  and  diarrhoea. 
After  a  calomel  purge  the  patient  was  better;  the  following  day,  how- 
ever, the  diarrhoea  started  up  again  and  the  temperature  rose.  The 
stools  were  numerous,  small,  containing  mucus  and  blood,  preceded 
by  pain  and  accompanied  by  tenesmus.  Many  of  the  stools  consisted 
of  nothing  but  blood  and  mucus.  Sixteen  movements  were  the  greatest 
number  recorded  in  a  day.  On  August  2d  ten  cubic  centimetres  of 
dysenteric  serum  were  injected.  There  seemed  to  be  some  improvement 
in  the  character  of  the  stools  following  the  injection.  On  August  4th 
and  6th  the  injections  were  repeated,  being  followed  each  time,  appar- 
ently, by  some  improvement  in  the  child's  condition.  The  blood  dis- 
appeared in  eight  or  nine  days,  and  the  child  had  then  five  movements 
daily,  consisting  largely  of  mucus. 

At  Hiker's  Island  a  number  of  men  were  filling  in  new  land.  Dysen- 
tery broke  out  and  spread  to  a  number  of  the  men,  as  well  as  to  the 
physician  in  charge.  Those  infected  had  usually  a  short,  sharp  attack 
with  a  quick  recovery.  Very  large  amounts  of  blood  were  passed  by 
some  of  the  sick. 

In  some  a  large  proportion  of  the  bacteria  isolated  were  bacilli  of 
the  Philippine  type.  No  other  type  of  dysentery  bacilli  was  found  in 
any  of  the  cases  in  this  epidemic. 

Charlton  and  Jehle  report  a  series  of  cases  occurring  in  St.  Ann's 
Hospital,  Vienna,  in  which  mannite  fermenting  types  were  the  only 
dysenteric-like  organisms  present.  The  cases  on  the  average  ran  a 
much  milder  course  than  those  in  whichtbe-  true  dysentery  bacilli  were 
present. 

When  the  agglutinating  characteristics  of  these  bacilli  and  their 
susceptibility  to  immune  sera  are  studied  carefully,  we  find  that  each 
of  the  three  types  differs  from  the  others.  The  mannite  and  the 
maltose  types,  since  in  animals  they  stimulate  abundant  common 
agglutinins  and  immune  bodies,  seem  more  closely  allied  to  each  other 
than  to  the  Shiga  type. 


THE  PARADYSENTERY  BACILLI 


259 


This  is  seen  in  the  following  tables,  in  which  bacilli  from  a  number 
of  cases  obtained  from  different  sources  are  tested  in  sera  from  animals 
which  have  each  received  a  single  type  of  dysentery  bacillus: 

TABLE  I. — Agghtination  of  bacilli  belonging  to  the  three  types  in  the  serum  of  a  young  goat 
injected  with  the  bacillus  isolated  by  Shiga,  in  Japan. 


Dilutions  of  Serum. 


Source.  1 : 20 
Type  I.    Shiga. 

1.  Original,  Japan— Shiga,  4--f 

2.  New  Haven— Duval,  4-4- 

3.  Tuckahoe— Carey,  4-  | 

4.  Coney  Island— Collins,  +4- 

5.  Mt.  Vernon,  Case  I.-Collins,    +4- 

6.  "          "         "    n.         "  4-4- 

7.  "  "         "III.         '•  +4- 


Type  II. 

8.  Original,  Mt.  Desert— Park,  + 

9.  New  York  City— Goodwin,  + 

10.  Hospital,  New  York— Collins,  + 

11.  Foundling  Hospital— Hiss,  + 

12.  Mt,  Vernon,  Case  I.— Collins,  + 

13.  "         "  "  II.         •«  +  + 

Type  III. 

14.  Original,  Manila— Flexner,  + 

15.  Baltimore— Duval,  -f  | 

16.  New  York  City— Wollstein,  +  + 

17.  Orange— Collins,  + 

18.  Hiker's  Island— Goodwin,  ++ 


1:100   1:200   1:500   1:2000  1:6000 


4-  + 


-f-f 


4-4- 
4-4- 
4-4- 


4-4- 
4-4- 
4-4- 

4-4- 
4-4- 
4-4- 


4-4- 


+-H 


4-1 
4-1 


The  serum  of  this  goat  before  injection  did  not  agglutinate  any  of  the  above  bacilli  in  1 : 10  dilution. 
+  -f  =  complete  reaction.  +  =  good  reaction.  |  =  slight  reaction. 

-I-  |  =  very  good  reaction.  ±  =  fair  reaction.  —  =  no  reaction. 


TABLE  II. — Showing  agglutination  of  members  of  three  types  in  the  serum  of  animals  injected 

with  bacilli  of  Type  II. 


Source. 
Type  I.    Shiga. 

1.  Japan— Shiga, 

2.  New  Haven— Duval, 

3.  Tuckahoe — Carey, 

jrypen. 

4.  Mt.  Desert— Park, 

ft.  Mt.  Vernon— Collins, 

6.  New  York— Hiss, 

Type  III. 

7.  Manila— Flexner, 

8.  Baltimore— Duval, 

9.  Hiker's— Goodwin, 


Goat  injected  with  No.  4. 


Rabbit  injected  with  No.  6. 


1:20      1:50      1:100    1:500    1:1000     1:20     1:50     1:100    1:500    1:800 


+  + 


+  + 
+-f 


+  + 


—  —        4-4- 


+  4- 


++ 


4-4-         —         — 


The  serum  of  the  above  animals  previous  to  immunization  did  not  agglutinate  any  of  the  above 
bacilli  in  a  1  :  20  dilution. 


260 


BACTERIA  PATHOGENIC  TO  MAN 


TABLE  III. — Showing  agglutinations  of  members  of  three  types  in  the  serum  of  animals 
injected  with  bacilli  of  Type  III. 

Rabbit  injected  with  Baltimore,  Duval. 

To  50  100  500          1000        5000      10,000 

Type  I. 
1.  Japan— Shiga,  and  5  other  cultures,     ++  +  + 

Typell. 
6.  Mt.  Desert— Park,  and  5  other  cultures,  +  +  +  +  + 

Type  III. 
Manila— Flexner,  and  5  other  cultures,  ++          ++          +  +          +  +       ++       ++          + 

Previous  to  immunization  the  serum  agglutinated  the  bacilli  of  Type  III.  in  1 :  20  dilution,  but 
none  of  the  others  even  in  1 : 10.  This  is  one  of  the  few  animals  in  which  agglutinins  for  Type  I. 
developed  through  the  injections  of  bacilli  of  the  other  types. 

TABLE  IV. — Showing  how  Type  III.  is  unable  to  absorb  the  agglutinins  produced  through 
injections  of  Type  II.     Serum  from  rabbit  inoculated  with  Mt.  Vernon  culture,  Type  II. 

Agglutinins  exhausted  with 


Type  I. 
Shiga,  5  other  cultures, 

Type  II. 

Mt.  Desert,  5  other  cultures, 
Type  III. 

Manila,  5  other  cultures, 


Serum 
before 
absorp- 
tion. 


Baltimore,  Duval. 


1  :  20      1  :  50     1  :  100    1  :  200    1  :  400 


Mt.  Vernon,  c.c.a 
T:  20      1 :  50     1 :  100 


1:10  —  —  —  —  —  —  —  — 


1:600      ++        ++ 


1  :  100         — 


+  + 


Before  injections  this  rabbit's  serum  agglutinated  Types  II.  and  III. 
in  1:20  dilutions. 

The  considerable  amount  of  common  agglutinins  affecting  Type  II. 
and  Type  III.  is  seen  to  be  absorbed  by  the  bacilli  of  either  type. 
The  larger  amount  of  specific  agglutinin  is  left  in  the  serum  when  any 
bacillus  other  than  one  of  identical  characteristics  with  the  bacillus 
used  in  the  immunization  is  employed. 


TABLE  V. — Showing  that  horse  injected  with  Shiga  and  Philippine  types  develop  specific 
agglutinins  for  the  bacilli  belonging  to  these  two  types  and  common  agglutinins  for  the 
.varieties  included  under  Type  II. 


•Cultures. 

Type  I. 

Shiga,  original,  and  4  others, 
'Type  II. 

Park,  original,  and  4  others, 
Type  II.    (B.) 

Brooklyn, 
Type  II.    (C.) 
Type  II.    (D.) 

Type  III. 
Flexner  original  and  4  others, 


Serum 
after 
injec- 
tions for 
several 
months. 


+600 

+600 
+300 
+600 

—1200 


Same  serum  after  saturation  with  cultures  of 


Shiga 
Type. 


Type  III.    Type  II. 


Pyocy- 
aneus. 


Typhoid.    Colon. 


+1500        —10 


—10 

+20 
—10 
—20 


+400 
—10 

+10 
—10 
—10 


+700        +1000 


—10 

+50 
+50 
+50 


+600 

+800 

+50 

+100 


+400        —10 


+500        +800 


+300 
+30 

+100 

+10 

+30 

+300 


+300 
+50 

+50 
+20 
+60 

+600 


The  manipulation  necessary  in  making  dilutions  and  filtering,  as  well 
as  the  effect  of  standing,  cause  a  certain  amount  of  destruction  of 
agglutinins. 


THE  PARADYSENTERY  BACILLI  261 

SIMMARY. — The  great  majority  of  the  bacilli  which  have  been 
isolated  from  cases  of  dysentery  not  due  to  amoebae,  and  which  must 
be  considered  as  being  exciting  factors  in  that  disease,  are  included  in 
three  distinct  varieties  or  types.  This  at  least  is  true  for  the  many 
cultures  which  we  have  isolated  or  obtained  from  others. 

The  type  most  frequently  found  in  severe  epidemics  is  that  of  the 
first  culture  isolated  by  Shiga.  Bacilli  identical  in  biochemical  and 
agglutinating  characteristics  with  this  bacillus  have  been  isolated  from 
cases  of  dysentery  in  many  parts  of  the  world.  None  of  the  bacilli 
belonging  to  this  type  produce  indol,  except,  perhaps,  in  a  trace,  or 
ferment  mannite,  maltose,  or  saccharose.  Animals  injected  with  this 
type  produce  specific  agglutinins  for  this  type  in  abundance  and  only 
very  little  that  combines  with  the  others. 

The  second  type  ferments  mannite  with  the  production  of  acid,  but 
does  not  split  maltose  or  saccharose  in  peptone  solution  or  agar. 

It  produces  indol.  Animals,  after  inoculations  with  it,  develop 
immune  bodies  and  agglutinins  specific  for  the  type.  They  also  develop 
in  considerable  proportion  immune  bodies  and  agglutinins  which  have 
affinity  for  the  bacilli  of  Type  III.  and  to  a  slight  extent  for  Type  I. 

The  third  type  is  nearest  to  the  colon  group,  since  it  not  only  produces 
indol  and  actively  ferments^mAHTiite,  but  also  acts  energetically  upon 
pure  maltose  and  feebly  upon  saccharose. 

Animals  injected  with  this  type  develop  specific  immune  bodies  and 
agglutinins,  and  also  abundant  immune  bodies  and  agglutinins  which 
have  an  affinity  for  the  bacilli  of  Type  II.  and  for  many  bacilli  of  the 
colon  group.  For  Type  I.  these  substances  are  but  slightly  developed. 

These  two  mannite  fermenting  types  are  widely  scattered  over  the 
world,  and  certainly  cause  characteristic  cases  and  epidemics  of  dysen- 
tery, although  on  the  average  the  disease  caused  by  them  is  milder 
than  when  due  to  the  Shiga  bacillus.  One  or  the  other  of  these  two 
types  also  appear  at  times  in  small  numbers  in  mixed  infections  where 
dysenteric  symptoms  are  almost  or  entirely  absent. 

Although  the  majority  of  bacilli  obtained  have  had  the  characteristics 
of  one  of  the  above  types,  still  bacilli  have  been  found  in  isolated  cases, 
.which,  although  agreeing  in  biochemical  characteristics  with  one  of 
the  three,  nevertheless  differed  in  producing  different  specific  agglutinins. 
A  few  bacilli  have  also  been  met  with  which  differ  slightly  in  biochemical 
as  well  as  agglutinating  characteristics. 

It  seems,  therefore,  that  these  three  types  should  be  considered  as 
the  characteristic  representatives  of  three  groups. 

In  consideration  of  all  the  above  facts,  it  seems  to  us  incorrect  to 
name  the  mannite-fermenting  groups  as  pseudodysentery  bacilli.  If 
we  call  them  all  dysentery  bacilli,  we  must  classify  them  as  dysentery 
bacilli  of  the  Shiga  group,  of  the  group  fermenting  mannite,  but  not 
maltose,  and  of  the  one  fermenting  both  mannite  and  maltose. 

This  manner  of  differentiating  the  groups  would  be  very  confusing, 
and  it  seems  to  us  more  convenient,  and  better,  to  restrict  the  name 
dysentery  to  bacilli  having  the  characteristics  of  the  bacillus  isolated 


262  BACTERIA  PATHOGENIC  TO  MAN 

by  Shiga,  and  give  the  name  paradysentery  to  the  other  two  groups 
which  approach  more  closely  the  colon  group  in  that  they  produce 
indol  and  have  a  greater  range  of  activity  in  fermenting  carbohydrates. 
An  additional  reason  for  the  use  of  the  prefix  para,  beyond  that  of 
convenience,  is  the  less  average  severity  of  the  disease  due  to  these 
types,  and  the  probability  that  there  will  be  found,  in  occasional  sporadic 
cases  and  epidemics  of  dysentery,  bacilli  which  have  a  causal  relation 
to  dysentery  and  exhibit  more  pronounced  characteristics  of  the  colon 
group  than  any  of  .the  varieties  so  far  isolated. 


CHAPTER  XX. 

THE  TYPHOID  BACILLUS  (BACILLUS  TYPHOSUS). 

THIS  organism  was  first  observed  by  Eberth,  and  independently  by 
Koch,  in  1880,  in  the  spleen  and  diseased  areas  of  the  intestine  in 
typhoid  cadavers,  but  was  not  obtained  in  pure  culture  or  its  principal 
biological  cultures  described  until  the  researches  of  Gaffky,  in  1884. 
The  methods  of  identification  employed  by  Gaffky  were  found  insuffi- 
cient to  separate  the  typhoid  bacillus  from  other  bacilli  of  the  colon- 
typhoid  group.  Every  known  cultural  characteristic  of  the  typhoid 
bacillus  was  found  to  be  duplicated  in  some  member  of  this  group,  and 
it  was  only  when  a  bacillus  combined  all  the  characteristics  of  a  typical 
variety  that  it  could  be  assumed  that  it  was  in  all  probability  the  typhoid 
bacillus.  The  absolute  identification  of  the  bacillus  only  became  pos- 
sible with  the  increase  of  our  knowledge  concerning  the  specific  immune 
substances  developed  in  the  bodies  of  immunized  animals.  Its  etiological 
relationship  to  typhoid  fever  has  been  particularly  difficult  of  demon- 
stration, for,  although  pathogenic  for  many  animals  when  subcutaneously 
or  intravenously  inoculated,  it  has  been  impossible  to  produce  infection 
in  the  natural  way  or  produce  gross  lesions  corresponding  closely  to 
those  occurring  generally  in  man.  It  has  been  recently  shown,  however, 
that  animals  under  certain  conditions,  when  their  power  of  resistance 
has  been  reduced,  may  be  rendered  susceptible  to  infection,  with  the 
production  of  more  or  less  characteristic  lesions.  These  results,  together 
with  the  specific  reactions  of  the  blood  serum  of  typhoid  patients,  the 
constant  presence  of  the  bacillus  typhosus  in  the  intestines  and  some 
of  the  organs  of  the  typhoid  cadavers,  the  frequent  isolation  of  this 
bacillus  from  the  roseola,  spleen,  blood,  and  excretions  of  the  sick 
during  life,  the  absence  of  the  bacilli  in  healthy  persons,  unless  they 
have  been  directly  exposed  to  or  are  convalescent  from  typhoid  infec- 
tion, all  these  have  demonstrated  scientifically  that  this  bacillus  is  the 
chief  etiological  factor  in  the  production  of  the  great  majority  of  cases 
designated  as  typhoid  fever. 

Morphology  and  Staining. — Typhoid  bacilli  are  short,  rather  plump 
rods  of  about  1,«  to  3/*  in  length  by  0.5^  to  0.8/*  in  diameter,  having 
rounded  ends,  and  often  growing  into  long  threads.  They  are  longer 
and  somewhat  more  slender  in  form  than  most  of  the  members  of  the 
colon  group  of  bacilli  (Figs.  88  and  89). 

The  typhoid  bacilli  stain  with  the  ordinary  aniline  colors,  but  a  little 
less  readily  than  do  most  other  bacteria.  Like  the  bacilli  of  the  colon 
and  paratyphoid  groups,  they  are  decolorized  by  Gram's  method. 


264 


BACTERIA  PATHOGENIC  TO  MAN 


Biology. — The  typhoid  bacillus  is  a  motile,  aerobic,  facultative  anae- 
robic, non-liquefying  bacillus.  It  develops  best  at  37°  C.;  over  40°  and 
below  30°  growth  is  retarded;  at  20°  it  is  still  moderate;  below  10° 
it  almost  ceases.  It  grows  slightly  more  abundantly  in  the  presence 
of  oxygen.  It  does  not  form  spores. 

RESISTANCE. — When  a  number  of  typhoid  bacilli  are  dried  most  of 
them  die  within  a  few  hours  and  a  few  frequently  remain  alive  for 
months,  but  sometimes  all  the  bacilli  die  very  quickly.  In  their 


FIG.  88 


FIG. 


Typhoid  bacilli  from  nutrient  agar. 
X  1100  diameters. 


Typhoid  bacilli  from  nutrient  gelatin. 
X  1100  diameters. 


resistance  to  heat  and  cold  they  behave  like  the  more  resistant,  non- 
spore-bearing  bacilli. 

Motility. — Typhoid  bacilli,  when  living  under  favorable  conditions, 
are  very  actively  motile,  the  smaller  ones  having  often  an  undulating 
motion,  while  the  larger  rods  move  about  rapidly.  In  different  cultures, 
however,  the  degree  of  motility  varies. 


FIG.  90 


FIG.  91 


J 


$ 


Flagella,  heavily  stained,  attached  to  bacilli. 


Typhoid  bacillus  with  stained  flagella. 


FLAGELLA. — These  are  often  numerous  and  spring  from  the  sides  as 
well  as  the  ends  of  the  bacilli,  but  many  short  rods  have  but  a  single 
terminal  flagellum  (Figs.  90  and  91). 


THE  TYPHOID  BACILLUS  l>(jo 

Cultivation. — Its  growth  on  most  sugar-free  culture  media  is  similar 
to  that  of  the  bacillus  coli  communis,  but  it  is  somewhat  slower  and 
not  quite  so  luxuriant. 

GROWTH  ON  GELATIN  PLATES  (Fig.  92). — The  colonies  growing 
deep  down  in  this  plate  medium  have  nothing  in  their  appearance  to 
distinguish  them;  they  appear  as  finely  granular  round  points  with  a 
sharp  margin  and  a  yellowish-brown  color.  The  superficial  colonies, 
however,  particularly  when  young,  are  often  quite  characteristic;  they 
are  transparent,  bluish-white  in  color,  with  an  irregular  outline,  not 
unlike  a  grape-leaf  in  shape.  Slightly  magnified  they  appear  homo- 
geneous in  structure,  but  marked  by  a  delicate  network  of  furrows. 
Surface  colonies  from  some  varieties  of  colon  bacilli  give  a  similar 
picture. 

FIG.  92 


A  superficial  colony  (1)  and  a  deep  colony  (2)  of  typhoid  bacilli  in  gelatin.    X  20  diameters. 

In  stick  cultures  in  gelatin  the  growth  is  mostly  on  the  surface,  appear- 
ing as  a  thin,  scalloped  extension,  which  gradually  reaches  out  to  the 
sides  of  the  tube.  In  the  track  of  the  needle  there  is  but  a  limited 
growth,  which  may  be  granular  or  uniform  in  structure,  and  of  a 
yellowish-brown  color.  There  is  no  liquefaction. 

GROWTH  IN  BOUILLON. — This  medium  is  uniformly  clouded  by  the 
typhoid  bacillus,  but  the  clouding  is  not  so  intense  as  by  the  colon 
bacillus.  When  the  bouillon  is  somewhat  alkaline  a  delicate  film  is 
sometimes  formed  on  the  surface  after  eighteen  to  twenty-four  hours' 
growth. 

GROWTH  ON  AGAR.— The  streak  cultures  on  agar  are  not  distinctive; 
a  transparent,  grayish  streak  is  formed. 

GROWTH  ON  POTATO. — The  growth  on  this  medium  was  formerly  of 
great  importance  in  identification,  but  now  other  media,  giving  more 
specific  characteristics,  have  been  discovered.  When  characteristic  the 
growth  is  almost  invisible,  but  luxuriant,  usually  covering  the  surface  of 


BACTERIA  PATHOGENIC  TO  MAN 

the  medium,  and  when  scraped  with  the  needle  offers  a  certain  resist- 
ance. In  some  cases,  however,  the  growth  is  restricted  to  the  immediate 
vicinity  of  the  point  of  inoculation.  Again,  the  growth  may  be  quite 
heavy  and  colored  yellowish-brown,  and  with  a  greenish  halo,  when 
it  is  very  similar  to  the  growth  of  the  colon  bacillus.  These  differences 
of  growth  on  potato  appear  to  be  chiefly  due  to  variations  in  the  sub- 
stance of  the  potato,  especially  in  its  reaction.  For  the  characteristic 
growth  the  potato  should  be  slightly  acid.  A  new  lot  of  potato  should 
always  be  tested  with  a  typical  typhoid  bacillus  as  a  control. 

INDOL  REACTION. — It  does  not  produce  indol.  This  test  was  pro- 
posed by  Kitasato  for  differentiating  the  typhoid  bacillus  from  other 
similar  bacilli,  such  as  those  of  the  colon  group,  which,  as  a  rule,  give 
the  indol  reaction. 

The  typhoid  bacillus,  like  the  colon  bacillus,  produces  alkaline 
substances  from  peptone. 

NEUTRAL  RED. — In  stick  cultures  in  glucose  agar  the  typhoid  bacillus 
produces  no  change,  while  the  colon  bacillus  decolorizes  the  medium 
and  produces  gas. 

EFFECT  OF  INHIBITING  SUBSTANCES  IN  CULTURE  FLUIDS. — The 
typhoid  bacillus  is  inhibited  by  weaker  solutions  of  formaldehyde, 
carbolic  acid,  and  other  disinfectants,  than  is  the  colon  bacillus.  Most 
typhoid-like  bacilli  resemble  the  typhoid  bacillus  in  this  respect. 

ACTION  ON  DIFFERENT  SUGARS. — The  determination  of  the  action 
upon  sugars  of  any  bacillus  belonging  to  the  typhoid  or  colon  group  is 
one  of  the  most  important  of  all  the  cultural  differential  tests.  It  has 
been  considered  in  detail  in  connection  with  the  colon  group. 

FERMENTATION. — While  the  typhoid  bacillus  does  not  ferment  glucose, 
galactose  and  levulose,  it  does  produce  acid  from  these  substances.  It 
evolves  gas  from  none  of  the  sugars. 

MILK. — The  typhoid  bacillus  does  not  cause  coagulation  when 
grown  in  milk.  In  litmus  whey  the  neutral  violet  color  becomes  more 
red  during  the  first  forty-eight  hours;  the  fluid,  however,  remains  clear. 

Production  of  Disease  in  Animals. — It  is  impossible  experimentally 
to  produce  true  typhoid  fever  in  animals.  Sickness  or  fatal  results 
without  the  appearance  of  the  typical  pathological  changes  have  regu- 
larly followed  animal  inoculations,  but  in  most  cases  they  could  easily 
be  traced  to  the  toxaemia  produced  by  the  substances  in  the  bodies  of 
the  bacilli  injected.  Typhoid  bacilli,  freshly  obtained  from  typhoid 
cases  and  introduced  subcutaneously  in  animals,  rapidly  die.  In  the 
peritoneal  cavity  they  may  increase,  causing  a  fatal  peritonitis  with 
toxic  poisoning.  By  accustoming  bacilli  to  the  animal  body  a  certain 
degree  of  increased  virulence  for  the  animal  can  be  obtained,  so  that 
smaller  amounts  of  culture  may  prove  fatal.  Among  the  most  success- 
ful efforts  in  this  direction  are  the  experiments  of  Cygnaeus  and  Seitz, 
who,  by  the  inoculation  of  typhoid  bacilli  into  dogs,  rabbits,  and  mice, 
produced  in  the  small  intestines  conditions  that  were  histologically  and 
to  the  naked  eye  analogous  to  those  found  in  the  human  subject. 
Their  results,  however,  were  not  constant. 


Till:  TYI'HOID  BACILLUS  267 

Experiments  indicate  that  the  presence  of  other  bacteria  in  the  body, 
and  of  exposure  to  the  effect  of  noxious  gases  in  lowering  the  natural 
resistance  of  the  individual,  render  him  more  susceptible  to  infection 
from  typhoid  fever. 

Distribution  of  Bacilli  in  the  Human  Subject.  Toxic  Effects. — 
Typhoid  fever  belongs  to  that  class  of  infectious  diseases  in  which  the 
specific  bacilli  may  occur  throughout  the  entire  circulation,  as  in  septi- 
caemia, or  remain  localized  in  certain  regions  in  the  body.  Wherever 
found  in  the  tissues  the  typhoid  bacilli  are  usually  observed  to  be 
arranged  in  groups  or  foci;  only  occasionally  are  they  found  singly. 
These  foci  are  formed,  most  probably,  during  life,  as  is  proved  by  the 
degenerative  changes  often  seen  about  them;  but  it  is  possible  that  the 
bacilli  may  also  multiply  somewhat  after  death. 

The  inflammatory  changes  in  the  lymphoid  tissue  and  other  cellular 
degenerations  so  often  found  in  typhoid  fever  in  the  internal  organs 
are  due  to  the  effects  of  the  soluble  toxic  substances  eliminated  by 
the  typhoid  bacilli.  The  inflammation  and  ulceration  of  Peyer's 
patches  are  the  central  feature,  these  being  more  directly  under  the 
influence  of  the  concentrated  bacterial  products.  In  typhoid  fever 
necrosis  of  the  tissues  of  the  internal  organs  is  of  comparatively  rare 
occurrence.  Caseation  of  the  mesenteric  glands,  which  is  commonly 
observed,  is  due  possibly  to  mixed  infection.  There  are,  however,  a 
number  of  cases  now  on  record  in  which  the  typhoid  bacillus  has  played 
the  part  of  pus  producer. 

Unusual  Location  of  Typhoid  Lesions  Occurring  as  Complications 
of  Typhoid  Fever. — Cases  of  sacculated  and  general  peritonitis,  sub- 
phrenic  abscess,  osteomyelitis,  periostitis,  and  inflammatory  procesess 
of  other  kinds  have  been  reported  as  being  due  to  the  typhoid  bacillus. 
Kruse  also  reports  an  abscess  of  the  spleen  which  contained  only  bacillus 
typhosus,  and  typhoid  abscess  of  the  liver  has  been  recorded  by  many. 
In  certain  cases  of  typhoid  pneumonia,  serous  pleurisy,  empyema,  and 
inflammations  of  the  brain  and  spinal  cord  or  their  membranes,  typhoid 
bacilli  exclusively  have  occurred.  The  inflammation  produced  may  or 
may  not  be  accompanied  by  the  formation  of  pus. 

Such  cases,  however,  are  of  comparatively  rare  occurrence,  because 
only  exceptionally  do  the  bacilli  mass  together  in  such  numbers  as 
to  become  pus  producers.  As  a  rule,  when  such  complications  occur 
in  typhoid  fever  they  are  due  to  secondary  or  mixed  infection  with 
the  staphylococcus,  pneumococcus,  streptococcus,  pyocyaneus,  and 
colon  bacillus.  Frequently  these  bacteria  are  found  side  by  side  with 
the  typhoid  bacilli;  in  such  cases  it  is  difficult  to  say  which  was  the 
primary  and  which  was  the  secondary  infection. 

The  distribution  of  the  typhoid  bacilli  in  different  parts  of  the  body 
is  explained  by  their  passage  through  the  circulation;  and  this  is  proved 
by  the  bacilli  being  found  in  the  earlier  days  of  the  disease  in  the  spleen 
constantly  and  frequently  in  the  blood  itself. 

The  typhoid  bacillus  can  be  transmitted  also  from  the  blood  of  the 
mother  to  the  foetus.  In  one  case  reported  by  Ernst  a  living  child,  four 


2G8  BACTERIA  PATHOGENIC  TO  MAN 

days  after  birth,  showed  evidences  of  general  typhoid  infection — icterus 
and  rose  spots. 

Not  infrequently  typhoid  bacilli  are  found  in  the  secretions.  They 
are  present  in  the  urine  in  about  20  per  cent,  of  the  cases  in  the  third 
and  fourth  weeks  of  typhoid  fever.  Slight  pathological  lesions  in  the 
kidneys  almost  always  occur  in  typhoid  fever,  but  severe  lesions  also 
sometimes  occur.  In  some  cases  the  urine  is  crowded  with  typhoid 
bacilli. 

In  cases  of  pneumonia  due  to  the  typhoid  bacillus  it  is  abundantly 
present  in  the  sputa,  and  care  should  be  taken  to  disinfect  the  expec- 
toration of  typhoid  patients.  In  typhoid  fever  the  bacilli  are  almost 
always  present  in  the  gall-bladder.  The  bacilli  are  usually  eliminated 
by  the  feces,  being  derived  from  the  ulcerated  portions  of  the  intestines ; 
their  growth  within  the  intestinal  contents  is,  with  few  exceptions,  not 
extensive. 

Not  only  do  the  very  great  majority  of  cases  examined  bacteriologically 
and  pathologically,  but  the  epidemiological  history  of  the  disease,  proves 
that  the  chief  mode  of  invasion  of  the  typhoid  bacillus  is  by  way  of 
the  mouth  and  stomach.  The  infective  material  is  discharged  principally 
by  means  of  the  excretions  and  secretions  of  the  sick — namely,  by  the 
feces,  the  urine,  and  occasionally  by  the  sputum. 

Occurrence  in  Healthy  Persons. — In  a  few  cases  they  have  been 
obtained  from  the  intestines  of  healthy  persons.  (Drigalski  and  Conradi,. 
Zeit.  Hyg.,  vol.  xxxix.  p.  283.) 

Duration  of  Life  in  Man. — The  bacilli  usually  disappear  from  the 
body  in  the  fourth  or  fifth  week,  but  may  remain  for  months  or  excep- 
tionally years  in  the  urine  and  in  the  gall-bladder.  They  have  been 
found  in  collections  of  pus  one  year  after  recovery  from  typhoid  fever. 

Duration  of  Life  Outside  of  the  Body. — It  is  of  importance  to  know 
for  what  length  of  time  the  typhoid  bacillus  is  capable  of  living  outside 
of  the  body;  but,  unfortunately,  owing  to  the  great  difficulties  in  proving 
the  presence  of  this  organism  in  natural  conditions,  our  knowledge  on 
this  point  is  still  incomplete.  In  feces  the  length  of  life  of  the  typhoid 
bacilli  is  very  variable,  depending  on  the  composition  of  the  feces  and 
of  the  varieties  of  bacteria  present;  sometimes  they  live  but  a  few  hours, 
usually  a  day,  exceptionally  for  very  long  periods.  Thus,  according  to 
I>evy  and  Kayser,  in  winter  typhoid  bacilli  may  remain  alive  in  feces 
for  five  months.  Foote  says  that  they  can  be  found  in  living  oysters 
for  a  month  at  a  time,  but  in  numerous  experiments  we  have  not  been 
able  to  find  them  after  five  days.  Their  life  in  privies  and  in  water, 
however,  is  usually  very  much  shorter.  As  a  rule,  they  can  be  detected 
in  river  water  no  longer  than  seven  days  after  introduction,  and  often 
not  after  forty-eight  hours.  The  less  contaminated  the  water,  the  longer 
the  bacilli  are  apt  to  live.  The  life  of  the  typhoid  bacillus  varies  accord- 
ing to  the  abundance  and  varieties  of  the  bacteria  associated  with  it, 
and  according  to  the  presence  or  absence  of  such  injurious  influences 
as  deleterious  chemicals,  high  temperature,  light,  desiccation,  etc.,  to 
which  it  is  peculiarly  sensitive.  Good  observers  claim  to  have  found 


THE  TYPHOID  BACILLUS  269 

bacilli  very  similar  to  typhoid  bacilli  in  the  soil  in  a  region  where  no 
typhoid  fever  was  known  to  exist. 

Communicability. — The  bacilli  may  reach  the  mouth  by  means  of 
infected  fingers  or  articles  of  various  kinds,  or  by  the  ingestion  of 
infected  food,  milk,  water,  etc.,  or  by  more  obscure  ways,  such  as  the 
eating  of  raw  oysters  and  clams  or  the  contamination  of  food  by  flies 
and  other  insects,  or  by  inhalation  through  the  mouth.  Of  the  greatest 
importance,  however,  is  the  production  of  infection  by  contaminated 
drinking-water  or  milk.  In  a  very  large  number  of  cases  indirect  proof 
of  this  mode  of  infection  has  been  afforded  by  finding  that  the  water 
had  been  contaminated  with  urine  or  feces  from  a  case  of  typhoid.  In 
a  few  instances  the  proof  has  been  direct — namely,  by  finding  typhoid 
bacilli  in  the  water.  Examples  of  infection  from  water  and  milk  have 
frequently  come  under  our  direct  observation.  The  following  instances 
may  be  cited :  A  large  force  of  workmen  obtained  their  drinking-water 
from  a  well  near  where  they  were  working.  Typhoid  fever  broke  out 
and  continued  to  spread  until  the  well  was  filled  up.  Investigation 
showed  that  some  of  the  sick,  in  the  early  stages  of  their  disease, 
repeatedly  infected  the  soil  surrounding  the  well  with  their  urine  and 
feces.  Another  example  occurred  in  which  typhoid  fever  broke  out 
along  the  course  of  a  creek  after  a  spring  freshet.  It  was  found  that, 
far  up  near  the  source  of  the  creek,  typhoid  feces  had  been  thrown  on 
one  of  its  banks  and  had  then  been  washed  into  the  stream. 

In  the  late  epidemic  at  Ithaca  some  1500  cases  developed  among 
those  using  the  infected  water  supply  of  the  town.  The  students  and 
towns-people  not  drinking  the  infected  supply  escaped. 

An  instance  of  milk  infection  secondary  to  water  infection  was  in 
the  case  of  a  milk  dealer  whose  son  came  home  suffering  from  typhoid 
fever.  The  feces  were  thrown  into  a  small  stream  which  ran  into  a 
pond  from  which  the  milk  cans  were  washed.  A  very  alarming  epidemic 
of  typhoid  developed,  which  was  confined  to  the  nouses  and  asylums 
supplied  with  this  milk.  During  the  Spanish-American  war  not  only 
water  infection  but  food  infection  was  noticed,  as  in  the  case  of  a  regi- 
ment where  certain  companies  were  badly  infected,  while  others  nearly 
escaped.  Each  company  had  its  separate  kitchen  and  food  supply,  and 
much  of  the  infection  could  be  traced  to  the  food,  the  contamination 
coming  through  the  flies.  Several  epidemics  have  been  traced  to 
oysters. 

Individual  Susceptibility. — In  this,  as  in  all  infectious  diseases, 
individual  susceptibility  plays  an  important  role  in  the  production  of 
infection.  Without  a  suitable  soil  upon  which  to  grow,  the  seed  cannot 
thrive.  There  must  in  many  be  some  disturbance  of  the  digestion, 
excesses  in  drinking,  etc.,  or  a  general  weakening  of  the  power  of  resist- 
ance of  the  individual,  caused  by  bad  food,  exposure  to  heat,  over- 
exertion,  etc.,  as  with  soldiers  and  prisoners,  for  example,  to  bring 
about  the  conditions  suitable  for  the  production  of  typhoid  fever. 

The  supposition  that  the  breathing  of  noxious  gases  predisposes  to 
the  disease,  though  possibly  true  to  a  certain  extent,  as  some  animal 


270  BACTERIA  PATHOGENIC  TO  MAN 

experiments  already  referred  to  would  seem  to  indicate,  has  not  yet 
been  conclusively  proven;  nor  do  Pettenkofer's  investigations  into  the 
relation  of  the  frequency  of  typhoid  fever  to  the  ground-water  level 
satisfactorily  explain  the  occurrence  of  the  disease  in  most  cases,  whether 
sporadically  or  in  epidemics. 

Immunization. — After  recovery  from  typhoid  fever  a  considerable 
immunity  is  present  which  lasts  for  years.  This  is  not  absolute,  as 
about  2  per  cent,  of  those  having  typhoid  fever  have  a  second  attack. 
This  attack  is  usually  a  mild  one.  Specific  immunization  against  experi- 
mental typhoid  infection  has  been  produced  in  animals  by  the  usual 
method  of  injecting  at  first  small  quantities  of  the  living  or  dead  typhoid 
bacilli  and  gradually  increasing  the  dose.  The  blood  serum  of  animals 
thus  immunized  has  been  found  to  possess  bactericidal  and  feeble  antitoxic 
properties  against  the  typhoid  bacillus.  These  characteristics  have  also 
been  observed  in  the  blood  serum  of  persons  who  are  convalescent  from 
typhoid  fever.  The  attempt  has  been  made  to  employ  the  typhoid 
serum  for  the  cure  of  typhoid  fever  in  man,  but  although  a  number  of 
individual  observers  have  reported  good  results  with  one  or  another  of 
the  sera  most  consider  that  little  or  no  good  is  derived  from  the  serum. 

VACCINATION  AGAINST  TYPHOID. — The  use  of  killed  typhoid  bacilli 
as  vaccines  has  been  advocated  by  Wright  and  tried  upon  some  8000 
persons  who  expected  to  be  subjected  to  danger  of  infection. 

About  2  mg.  of  an  agar  tube  culture  which  had  been  suspended  in 
bouillon  and  heated  was  used  at  first,  but  now  0.3  to  0.1  c.c.  of  a  bouillon 
culture  according  to  the  density  of  suspension  is  heated  to  60°  C.  for  five 
minutes.  For  a  day  or  two  the  injection  produces  a  slight  fever  and  local 
pain,  followed  in  a  few  days  by  the  development  of  bactericidal  substances 
in  the  blood,  apparently  sufficient  in  amount  to  give  some  immunity 
lasting  for  a  year  or  more.  A  second  injection  adds  to  the  degree  of 
immunity.  In  49,600  individuals  under  observation  in  India  and  Africa, 
8600  were  thus  treated.  The  disease  appeared  in  them  to  the  extent  of 
2.25  per  cent.,  with  a  case  mortality  of  12  per  cent.  In  the  41,000 
uninoculated  there  was  a  case  percentage  of  5.75  per  cent.,  and  a 
mortality  of  26  per  cent.  The  use  of  a  protective  serum,  or,  when 
this  cannot  be  obtained,  of  dead  cultures,  would,  therefore,  seem  to  be 
advisable  where  great  danger  of  typhoid  infection  exists. 

Diagnosis  by  Means  of  the  Widal  or  Serum  Reaction. 

The  chief  practical  application  of  our  knowledge  of  the  specific 
substances  developed  in  the  blood  of  persons  sick  with  typhoid  fever 
has  been  as  an  aid  to  diagnosis. 

In  1894-95  Pfeiffer  showed  that  when  cultures  containing  dead  or 
living  cholera  spirilla  or  typhoid  bacilli  are  injected  subcutaneously 
into  animals  or  man,  specific  protective  substances  are  formed  in  the 
blood  of  the  individuals  thus  treated.  These  substances  confer  a  more 
or  less  complete  immunity  against  the  invasion  of  the  living  germs  of 
the  respective  diseases.  He  also  described  the  occurrence  of  a  peculiar 


THE  TYPHOID  BACILLUS  071 

phenomenon  when  some  fresh  culture  of  the  typhoid  bacillus  on  agar 
is  added  to  a  small  quantity  of  serum  from  an  animal  immunized 
against  typhoid  bacilli  and  the  mixture  injected  into  the  peritoneal  cavity 
of  a  non-immunized  guinea-pig.  After  this  procedure,  if  from  time  to 
time  minute  drops  of  the  liquid  be  withdrawn  in  a  capillary  tube  and 
examined  microscopically,  it  is  found  that  the  bacteria, "  previously 
motile  and  vigorous  and  which  remain  so  in  control  animals  inoculated 
without  the  specific  serum,  rapidly  lose  their  motility  and  die.  They 
are  first  immobilized,  then  they  become  somewhat  swollen  and  agglom- 
erated into  balls  or  clumps,  which  gradually  become  paler  and  paler, 
until  finally  they  are  dissolved  in  the  peritoneal  fluid.  This  process 
usually  takes  place  in  about  twenty  minutes,  provided  a  sufficient 
degree,  of  immunity  be  present  in  the  animals  from  which  the  serum 
was  obtained.  The  animals  injected  with  the  mixture  of  the  serum  of 
immunized  animals  and  typhoid  cultures  remain  unaffected,  while 
control  animals  treated  with  a  fluid  containing  only  the  serum  of  non- 
immunized  animals  mixed  with  typhoid  cultures  die.  Pfeiffer  claimed 
that  the  reaction  of  the  serum  thus  employed  is  so  distinctly  specific 
that  it  could  serve  for  the  differential  diagnosis  of  the  cholera  vibrion 
or  typhoid  bacillus  from  other  vibrions  or  allied  bacilli,  such  as  Finkler's 
and  Prior's  or  those  of  the  colon  group. 

In  March,  1896,  Pfeiffer  and  Kolle  published  an  article  entitled 
"The  Differential  Diagnosis  of  Typhoid  Fever  by  Means  of  the  Serum 
of  Animals  Immunized  against  Typhoid  Infection,"  in  which  they 
claimed  that  by  the  presence  or  absence  of  this  reaction  in  the  serum 
of  convalescents  from  suspected  typhoid  fever  the  nature  of  the  dis- 
ease could  be  determined.  It  was  further  found,  if  the  serum  of  an 
animal  thoroughly  immunized  to  the  typhoid  bacillus  was  diluted 
with  40  parts  of  bouillon,  and  a  similar  dilution  made  of  the  serum  of 
non-immunized  animals,  and  both  solutions  were  then  inoculated  with 
a  culture  of  the  typhoid  bacillus  and  placed  in  the  incubator  at  37°  C., 
that  after  the  expiration  of  one  hour  macroscopic  differences  in  the 
culture  could  be  observed,  which  increased  in  distinctness  for  four 
hours  and  then  gradually  disappeared.  The  reaction  occurring  is 
described  as  follows:  In  the  tubes  in  which  the  typhoid  culture  is 
mixed  with  typhoid  serum  the  bacilli  are  agglomerated  in  fine,  whitish 
flakes,  which  settle  to  the  bottom  of  the  tube,  while  the  supernatant 
fluid  is  clear  or  only  slightly  cloudy.  On  the  other  hand,  the  tubes 
containing  mixtures  of  bouillon  with  cholera  or  coli  serum,  or  the 
serum  of  non-immunized  animals  inoculated  with  the  typhoid  bacilli, 
become  and  remain  uniformly  and  intensely  cloudy.  These  serum 
mixtures,  examined  microscopically  in  a  hanging  drop,  show  distinct 
differences.  The  typhoid  serum  mixture  inoculated  with  the  typhoid 
bacilli  exhibits  the  organisms  entirely  motionless,  lying  clumped  together 
in  heaps;  in  the  other  mixtures  the  bacilli  are  actively  motile. 

Similar  observations  were  made  independently  by  Grul>er  and 
Durham,  who  maintained,  however,  that  the  reaction  described  by 
Pfeiffer  was  by  no  means  specific,  and  that  when  the  reaction  is  positive 


272  BACTERIA  PATHOGENIC  TO  MAN 

the  diagnosis  still  remains  in  doubt,  for  the  reaction  is  quantitative  only, 
and  not  qualitative.  They  concluded,  nevertheless,  that  these  investiga- 
tions would  render  valuable  assistance  in  the  clinical  diagnosis  of  cholera 
and  typhoid  fever. 

Gruber-Widal  Test. — The  first  application  of  the  use  of  serum,  how- 
ever, for  the  early  diagnosis  of  typhoid  fever  on  a  more  extensive 
scale  was  made  by  Widal,  and  reported  with  great  fulness  and  detail 
in  a  communication  published  in  June,  1896.  Widal  confirmed  the 
reaction  as  above  described,  proved  that  the  agglutinative  reaction 
usually  occurred  early,  elaborated  the  test,  and  proposed  a  method  by 
which  it  could  be  practically  applied  for  diagnostic  purposes.  Since 
then  the  serum  test  for  the  diagnosis  of  typhoid  fever  has  come  into 
general  use  in  bacteriological  laboratories  in  all  parts  of  the  world,  and 
though  the  extravagant  expectations  raised  at  the  time  when  Widal 
first  announced  his  method  of  applying  this  test  have  not  been  entirely 
fulfilled,  it  has,  nevertheless,  proved  to  be  of  great  assistance  in  the 
diagnosis  of  obscure  cases  of  the  disease,  and  is  now  one  of  the  recog- 
nized tests  for  the  differentiation  of  the  typhoid  bacillus. 

It  should  also  be  mentioned  that  to  Wyatt  Johnson,  of  Montreal, 
belongs  the  credit  of  having  brought  this  test  more  conspicuously  before 
the  public,  by  introducing  its  use  into  municipal  laboratories,  suggesting 
that  dried  blood  should  be  employed  in  place  of  blood  serum  (Widal 
having  previously  noticed  that  drying  did  not  destroy  the  agglutinating 
properties  of  typhoid  blood) ;  and  that  in  October,  1896,  the  serum  test 
was  regularly  introduced  in  the  New  York  Department  of  Health  Labo- 
ratory for  the  routine  examination  of  the  blood  serum  of  suspected 
cases  of  typhoid  fever.  Since  then  numerous  health  departments  have 
followed  the  example  set  by  those  of  Montreal  and  New  York. 

Use  of  Dried  Blood.  DIRECTIONS  FOR  PREPARING  SPECIMENS  OF 
BLOOD. — The  skin  covering  the  tip  of  the  finger  or  the  ear  is  thoroughly 
cleansed,  and  is  then  pricked  with  a  needle  deeply  enough  to  cause 
several  drops  of  blood  to  exude.  Two  fair-sized  drops  are  then  placed 
on  a  glass  slide,  one  near  either  end,  and  allowed  to  dry.  Glazed  paper 
may  also  be  employed,  but  it  is  not  as  good,  for  the  blood  soaks  more 
or  less  into  it,  and  later,  when  it  is  dissolved,  some  of  the  paper  fibre  is 
apt  to  be  rubbed  off  with  it.  The  slide  is  placed  in  a  box  for  protection. 

PREPARATION  OF  SPECIMEN  OF  BLOOD  FOR  EXAMINATION. — In  pre- 
paring the  specimens  for  examination  the  dried  blood,  if  accuracy  is 
desired,  is  first  weighed  and  then  brought  into  solution  by  adding  to 
it  and  mixing  it  with  nine  times  the  quantity  of  water;  then  a  minute 
drop  of  this  decidedly  reddish  mixture  is  placed  on  a  cover-glass,  and 
to  it  is  added  a  similar  drop  of  an  eighteen  to  twenty-four-hour-old 
bouillon  culture  of  the  typhoid  bacillus,  which,  if  it  has  a  slight  pellicle, 
should  be  well  shaken.  The  drops,  after  being  mixed,  should  have  a 
faint  reddish  or  pink  tinge  in  this,  the  most  highly  concentrated  serum 
dilution.  Higher  dilutions  are  prepared  by  adding  sterile  broth,  water, 
or  salt  solution  to  the  1 : 5  blood  mixture.  The  cover-glass  with  the 
mixture  on  the  surface  is  inverted  over  a  hollow  slide  (the  edges  about 


THE  TYPHOID  BACILLUS  273 

the  concavity  having  been  carefully  smeared  with  vaselin,  so  as  to  make 
a  closed  chamber),  and  the  hanging  drop  then  examined  under  the 
microscope  by  either  daylight  or  artificial  light,  a  high-power  dry  lens 
being  used,  or,  somewhat  less  serviceably,  a  1/12  oil-immersion  lens. 
Ordinarily  the  dried  blood  is  not  weighed,  but  the  measure  of  dilution 
is  estimated  by  the  color  of  the  drop.  To  judge  this  the  beginner 
must  carefully  make  dilutions  of  fluid  blood  and  notice  the  depth  of 
color  in  1:10  and  1 : 20  dilutions.  Besides  the  faulty  judgment  of  the 
dilution  color  by  the  examiner,  the  variation  in  depth  of  color  of 
different  specimens  of  blood  makes  the  estimation  of  dilutions  more 
or  less  inaccurate,  but  fortunately  this  does  not  greatly  interfere  with 
the  value  of  the  test. 

The  Reaction. — If  the  reaction  takes  place  rapidly  the  first  glance 
through  the  microscope  reveals  the  reaction  almost  completed,  most  of 
the  bacilli  being  in  loose  clumps  and  nearly  or  altogether  motionless 


FIG.  93 


Gruber-Widal  reaction.    Bacilli  gathered  into  one  large  and  two  small  clumps,  the  few  isolated 
bacteria  being  motionless  or  almost  so. 

(Fig.  93).  Between  the  clumps  are  clear  spaces  containing  few  or  no 
isolated  bacilli.  If  the  reaction  is  a  little  less  complete  a  few  bacilli 
may  be  found  moving  slowly  between  the  clumps  in  an  aimless  way, 
while  others  attached  to  the  clumps  by  one  end  are  apparently  trying 
to  pull  away,  much  as  a  fly  caught  on  fly-paper  struggles  for  freedom. 
If  the  agglutinating  substances  are  present,  but  still  less  abundant,  the 
reaction  may  be  watched  through  the  whole  course  of  its  development. 
Immediately  after  mixing  the  blood  and  culture  together  it  will  be 
noticed  that  the  bacilli  move  more  slowly  than  before  the  addition  of 
serum.  Some  of  these  soon  cease  all  progressive  movement,  and  it  will 
be  seen  that  they  are  gathering  together  in  small  groups  of  two  or  more, 
the  individual  bacilli  being  still  somewhat  separated  from  each  other. 
Gradually  they  close  up  the  spaces  between  them,  and  clumps  are 

18 


274  BACTERIA  PATHOGENIC  TO  MAN 

formed.  According  to  the  completeness  of  the  reaction,  either  all  of 
the  bacilli  may  finally  become  clumped  and  immobilized  or  only  a  small 
portion  of  them,  the  rest  remaining  freely  motile,  and  those  clumped 
may  appear  to  be  struggling  for  freedom.  With  blood  containing  a 
large  amount  of  agglutinating  substances  all  the  gradations  in  the 
intensity  of  the  reaction  may  be  observed,  from  those  shown  in  a 
marked  and  immediate  reaction  to  those  appearing  in  a  late  and  in- 
definite one,  by  simply  varying  the  proportion  of  blood  added  to  the 
culture  fluid. 

PSEUDOREACTIONS. — If  too  concentrated  a  solution  of  dried  blood 
from  a  healthy  person  is  employed  a  picture  is  often  obtained  which 
may  be  mistaken  for  a  reaction.  Dissolved  blood  always  shows  a  vary- 
ing amount  of  detritus,  partly  in  the  form  of  fibrinous  clumps;  and 
prolonged  microscopic  examination  of  the  mixture  of  dissolved  blood 
with  a  culture  fluid  shows  that  the  bacilli,  inhibited  by  substances  in  the 
blood,  often  become  more  or  less  entangled  in  these  clumps,  and  in 
the  course  of  one-half  to  one  hour  very  few  isolated  motile  bacteria  are 
seen.  The  fibrinous  clumps  alone,  especially  if  examined  with  a  poor 
light  by  a  beginner,  may  be  easily  mistaken  for  clumps  of  bacilli.  Again, 
the  bacilli  may  become  fixed  after  remaining  for  one-half  to  two  hours, 
by  slight  drying  of  the  drop  or  the  effect  of  substances  on  the  cover- 
glass.  The  reaction  in  typhoid  is  chiefly  due  to  specific  substances, 
but  clumping  and  inhibition  of  movement  similar  in  character  may  be 
caused  by  other  substances  such  as  exist  in  normal  horse  and  other 
serums.  This  is  a  very  important  fact  to  keep  in  mind.  (For  details 
of  technique  see  pages  81-83.) 

In  pseudoreactions  Wilson  has  noticed  that  many  free  bacilli  are 
apt  to  be  gathered  at  the  margin  of  the  hanging  drop. 

Use  of  Serum.  MODE  OF  OBTAINING  SERUM  FOR  EXAMINATION.— 
Fluid  blood  serum  can  easily  be  obtained  in  two  ways :  First,  the  serum 
may  be  obtained  directly  from  the  blood,  thus :  The  tip  of  the  finger 
or  ear  is  pricked  with  a  lancet-shaped  needle,  and  the  blood  as  it  issues 
is  allowed  to  fill  by  gravity  a  capillary  tube  having  a  central  bulb.  The 
ends  of  the  tube  are  then  sealed  by  heat  or  melted  wax,  or  candle  grease, 
and  as  the  blood  clots  a  few  drops  of  serum  separate.  To  obtain  larger 
amounts  of  serum  for  a  microscopic  examination  the  blood  is  milked 
out  from  the  puncture  into  a  small  homoeopathic  vial  or  test-tube.  One 
cubic  centimetre  of  blood  can  easily  be  collected  in  this  way.  The 
vial  is  then  corked  and  placed  on  the  ice  to  allow  the  serum  to  sepa- 
rate. As  a  rule  one  or  two  drops  of  serum  are  obtainable  at  the  end 
of  three  or  four  hours.  Second,  the  serum  may  be  obtained  from 
blisters.  This  gives  more  serum,  but  causes  twelve  hours'  delay.  The 
method  is  as  follows :  A  section  of  cantharides  plaster,  the  size  of  a 
5-cent  piece,  is  applied  to  the  skin  at  some  spot  on  the  chest  or  abdo- 
men. A  blister  forms  in  from  six  to  eighteen  hours.  This  should  be 
protected  from  injury  by  a  vaccine  shield  or  bunion  plaster.  The  serum 
from  the  blister  is  collected  in  a  capillary  tube,  the  ends  of  which  are 
then  sealed.  Several  drops  of  the  serum  can  easily  be  obtained  from  a 


THE  TYPHOID  HAC/LLUS  275 

blister  so  small  that  it  is  practically  painless  and  harmless.  The  serum 
obtained  is  clear  and  admirably  suited  for  the  test. 

ADVANTAGES  AND  DISADVANTAGES  OF  SERUM,  DRIED  BLOOD  AND 
FLUID  BLOOD  FOR  THE  SERUM  TEST.— The  dried  blood  is  easily  and 
quickly  obtained,  and  does  not  deteriorate  or  become  contaminated  by 
bacterial  growth.  It  is  readily  transported,  and  seems  to  be  of  nearly 
equal  strength  with  the  serum  in  its  agglutinating  properties.  It  must 
in  use,  however,  be  diluted  with  at  least  five  times  its  bulk  of  water, 
otherwise  it  is  too  viscid  to  be  properly  employed.  The  amount  of 
dilution  can  only  be  determined  roughly  by  the  color  of  the  resulting 
mixture,  for  it  is  impossible  to  estimate  accurately  the  amount  of  dried 
blood  from  the  size  of  the  drop,  and  it  is  too  much  trouble  to  weigh  it 
accurately.  Serum,  on  the  other  hand,  can  be  used  in  any  dilution 
desired,  varying  from  a  mixture  which  contains  equal  parts  of  serum 
and  broth  culture  to  that  containing  1  part  of  serum  to  100  parts  of 
culture  or  more,  and  this  can  be  exactly  measured  by  a  graduated  pipette, 
or,  roughly,  by  a  measured  platinum  loop.  The  disadvantages  in  the  use 
of  serum  are  entirely  due  to  the  slight  difficulty  in  collecting  and  trans- 
porting it,  and  the  delay  in  obtaining  it  when  a  blister  is  employed.  If 
the  serum  is  obtained  from  blood  after  clotting  has  occurred  a  greater 
quantity  of  blood  must  be  drawn  than  is  necessary  when  the  dried-blood 
method  is  used;  if  it  is  obtained  from  a  blister,  a  delay  of  six  to  eighteen 
hours  is  required.  The  transportation  of  the  serum  in  capillary  tubes 
presents  no  difficulties  if  tubes  of  sufficiently  thick  and  tough  glass  are 
employed  and  placed  in  tiny  wooden  boxes.  For  scientific  investiga- 
tions and  for  accurate  results,  particularly  in  obscure  cases,  the  use  of 
fluid  serum  is  to  be  preferred  to  dried  blood.  Practically,  however,  the 
results  are  nearly  as  good  for  diagnostic  purposes  from  the  dried  blood 
as  from  the  serum. 

FLUID  BLOOD. — When  properly  obtained  this  gives  good  results. 
The  Thoma-Zeiss  blood  pipette  is  very  useful.  Lance  finger-tip  or 
ear  and  draw  the  blood  into  the  pipette  to  the  mark  0.5.  Then  distilled 
wafer  is  sucked  up  in  sufficient  amount  to  make  the  desired  solution. 
One  loop  of  this  is  added  to  one  loop  of  bouillon  culture. 

THE  CULTURE  TO  BE  EMPLOYED. — It  is  important  that  the  culture 
employed  for  serum-tests  should  be  a  suitable  one,  for  although  all 
cultures  show  the  reaction,  yet  some  respond  much  better  and  in 
higher  dilutions  than  others.  Cultures  freshly  obtained  from  typhoid 
cases  are  not  as  sensitive  as  those  grown  for  some  time  on  nutrient 
media.  Decrease  in  virulence  is  apt  to  be  accompanied  by  increase  of 
capacity  for  agglutination.  For  the  past  seven  years  we  have  used  a 
culture  obtained  from  Pfeiffer.  A  broth  culture  of  the  typhoid  bacillus 
developed  at  25°  to  35°  C.,  not  over  twenty-four  hours  old,  in  which  the 
bacilli  are  isolated  and  actively  motile,  has  been  found  to  give  us  the 
most  satisfactory  results.  Cultures  grown  at  temperatures  over  38°  C. 
are  not  apt  to  agglutinate  so  well  as  those  grown  at  lower  temperatures. 
Stock  cultures  of  typhoid  bacilli  can  be  preserved  on  nutrient  agar  in 
sealed  tubes  and  kept  in  the  ice-box.  These  remain  alive  for  months  or 


/  276  BACTERIA  PATHOGENIC  TO  MAN 

even  years.     From  time  to  time  one  of  these  is  taken  out  and  used  to 
§tart  a  fresh  series  of  bouillon  cultures. 

.DILUTION  OF  THE  BLOOD  SERUM  TO  BE  EMPLOYED  AND  TIME 
INQUIRED  FOR  THE  DEVELOPMENT  OF  REACTION. — The  serum  test, 
as  has  been  pointed  out,  is  quantitative  and  not  qualitative.  By  this 
it  is  not  meant  to  assert  that  all  the  agglutinating  substances  produced 
/  in  the  blood  of  a  patient  suffering  from  typhoid  infection  are  the  same 
/'as  those  present  in  small  amount  in  normal  blood,  or  those  produced 
'in  the  blood  of  persons  sick  from  other  infections.  It  is  true,  however, 
that  the  apparent  effect  upon  the  bacilli  is  identical,  the  difference 
being  that  in  typhoid  fever,  as  a  rule,  substances  which  cause  this 
reaction  are  usually  far  in  excess  of  the  amount  which  ever  appears  in 
non-typhoid  blood,  so  that  the  reaction  occurs  after  the  addition  to  the 
culture  of  far  smaller  quantities  of  serum  than  in  other  diseases,  or 
when  the  same  dilution  is  used  it  occurs  far  more  quickly  and  completely 
with  the  typhoid  serum.  The  agglutinins  which  develop  in  animals 
after  immunization  with  many  bacilli  comprise  those  which  are  specific 
and  those  which  have  affinity  fcg.,  widely  differing  varieties.  (See 
chapter  on  Agglutinins.)  It  is  most  important  to  remember  that  it  is 
purely  a  matter  of  experience  tp  determine  in  any  type  of  infection 
what  degree  of  agglutinating^treng^h  in  a  serum  is  of  diagnostic 
value. 

The  results  obtained  in  the  Health  Department  Laboratories,  as 
well  as  elsewhere,  have  shown  that  in  a  certain  proportion  of  cases 
not  typhoid  fever  a  slow  reaction  occurs  in  a  1 : 10  dilution  of 
serum  or  blood;  but  very  rarely  does  a  complete  reaction  occur  in! 
this  dilution  within  fifteen  minutes.  When  dried  blood  is  used  the 
slight  tendency  of  non-typhoid  blood  in  1 : 10  dilution  to  produce 
agglutination  is  increased  by  the  presence  of  the  fibrinous  clumps, 
.and  perhaps  by  other  substances  derived  from  the  disintegrated  blood 
cells. 

From  many  cases  examined  it  has  been  found  that  in  dilutions] 
of  1 : 20  a  quick  reaction  is  almost  never  produced  in  any  febrile  dis- 
ease other  than  that  due  to  typhoid  or  paratyphoid  bacillus  infection,! 
while  in  typhoid  fever  such  a  distinct  reaction  often  occurs  with  dilu-j 
lions  of  1: 100  or  more.  It  is  possible  that  some  cases  of  paratyphoid; 
infection  give  a  prompt  reaction  in  1 : 20  dilutions,  but  if  this  is  so,  it 
is  not  a  serious  drawback. 

The  mode  of  procedure,  therefore,  as  now  employed  is  as  follows: 
The  test  is  first  made  with  the  typhoid  bacillus  in  a  5  per  cent,  solution  [ 
•of  serum  or  blood.  In  the  case  of  serum,  one  part  of  a  1 : 10  dilution  isj 
added  to  one  of  the  bouillon  culture.  With  dried  blood,  a  solution  of] 
the  blood  is  first  made,  and  the  dilution  guessed  from  the  color.  Toj 
obtain  an  idea  of  the  dilution  by  the  color,  known  amounts  of  blood 
are  dried  and  then  mixed  with  definite  amounts  of  water;  the  colors 
resulting  are  fixed  in  the  memory  as  guides  for  future  tests.  If  there  isj 
no  reaction — that  is  to  say,  if  within  five  minutes  no  marked  change] 
is  noted  in  the  motility  of  the  bacilli,  and  no  clumping  occurs — nothing; 


777 /•;   TYriluID  I!. \CILLUS  277 

more  is  needed;  the  result  is  negative.  If  marked  clumping  and 
immobilization  of  the  bacilli  immediately  begin  and  become  complete 
within  five  minutes,  this  is  termed  a  marked  immediate  typhoid 
reaction,  and  no  further  test  is  considered  necessary,  though  it  is 
always  advisable  to  confirm  the  reaction  with  higher  dilutions  up  to 
1:50  or  more,  so  as  to  measure  the  exact  strength  of  the  reaction.  If 
in  the  1 : 20  dilution  a  complete  reaction  takes  place  within  thirty 
minutes,  the  blood  is  considered  to  have  come  from  a  case  of  typhoid 
infection,  while  if  a  less  complete  reaction  occurs  it  is  considered  that 
only  a  probability  of  typhoid  infection  has  been  established.  By  many 
the  time  allowed  for  the  development  of  the  reaction  with  the  high 
dilutions  is  from  one  to  two  hours,  but  to  us  thirty  minutes  with  the 
comparatively  low  dilution  of  1 : 20  seems  safer  and  more  convenient. 
Positive  results  obtained  in  this  way  may  be  considered  conclusive, 
unless  there  be  grounds  for  suspecting  that  the  reaction  may  be  due 
to  a  previous  fairly  recent  attack.  In  our  opinion  the  failure  of  the 
reaction  in  one  examination  by  no  means  excludes  the  presence  of 
typhoid  infection.  If  the  case  clinically  remains  doubtful,  the  examina- 
tion should  be  repeated  within  a  few  days. 

USE  OF  DEAD  CULTURES. — Properly  killed  typhoid  bacilli  respond 
well  to  the  agglutination  test.  For  the  physician  at  his  office  the 
dead  bacilli  offer  many  advantages.  The  reaction  is  slower  than 
with  the  living  cultures  and  is  observed  either  macroscopically  or 
microscopically.  A  number  of  firms  now  supply  outfits  for  the  serum 
test.  These  outfits  consist  of  a  number  of  small  tubes  containing  an 
emulsion  of  dead  typhoid  bacilli.  Directions  accompany  the  outfit. 

PROPORTION  OF  CASES  OF  TYPHOID  FEVER  IN  WHICH  A  DEFINITE 
REACTION  OCCURS,  AND  THE  TIME  OF  ITS  APPEARANCE. — As  the  result 
of  a  large  number  of  cases  examined  in  the  Health  Department  Labo- 
ratories, it  was  found  that  about  20  per  cent,  gave  positive  results 
in  the  first  wreek,  about  60  per  cent,  in  the  second  week,  about  80  per 
cent,  in  the  third  week,  about  90  per  cent,  in  the  fourth  week,  and 
about  75  per  cent,  in  the  second  month  of  the  disease.  In  88  per 
cent,  of  the  cases  in  which  repeated  examinations  were  made  (hospital 
cases)  a  definite  typhoid  reaction  was  present  at  some  time  during  the 
illness. 

PERSISTENCE  OF  THE  REACTION. — In  persons  who  have  recovered 
from  typhoid  fever  this  peculiar  property  of  the  blood  serum  may 
persist  for  a  number  of  months.  Thus  a  definite  typhoid  reaction  has 
been  observed  from  three  months  to  a  year  after  convalescence, 
and  a  slight  reaction,  though  much  less  than  sufficient  to  establish  a 
diagnosis  of  typhoid  infection,  from  one  to  fifteen  years  after  the  disease. 

REACTION  WITH  THE  BLOOD  SERUM  OF  HEALTHY  PERSONS  AND 
OF  THOSE  ILL  WITH  DISEASES  OTHER  THAN  TYPHOID  FEVER. — In  the 
blood  serum  of  over  one  hundred  healthy  persons  examined  in  the 
Health  Department  Laboratories  an  immediate  marked  reaction  has  not 
been  observed  in  a  1 : 10  dilution.  In  several  hundred  cases  of  diseases, 
eventually  not  believed  by  the  physicians  in  charge  to  be  typhoid 


278  BACTERIA  PATHOGENIC  TO  MAN 

fever,  only  very  rarely  did  the  serum  give  a  marked  immediate  reac- 
tion in  a  1 : 10  dilution.  In  the  light  of  past  experience,  I  believe  a 
typhoid  or  paratyphoid  infection,  though  not  a  typical  typhoid  fever, 
to  have  existed  in  these  cases.  These  results  have  been  confirmed  by 
others,  the  question  of  dilution  having  recently  been  made  the  subject  of 
elaborate  investigations,  with  the  view  of  determining,  if  possible,  at 
what  dilution  the  typhoid  serum  would  react  while  others  would  not. 
Thus,  Schultz  reports  that  among  100  cases  of  non-typhoid  febrile 
diseases  apparently  positive  results  were  obtained  in  19  with  dilutions 
of  1:5,  in  11  of  these  with  1:10,  in  7  with  1:15,  in  3  with  1 : 20,  and  in 
1  a  very  faint  reaction  with  1:25;  whereas,  in  as  many  cases  of  true 
typhoid  he  never  failed  with  dilutions  of  1 : 50.  In  these  experiments 
it  must  be  noted,  however,  that  the  time  limit  was  from  one  to  two 
hours.  A  faint  reaction  with  a  1 : 25  dilution  with  a  time  limit  of  two 
hours  indicates  less  agglutinating  substance  than  an  immediate  com- 
plete reaction  with  a  1 : 10  dilution. 

From  an  experience  with  the  practical  application  of  the  serum  test 
for  the  diagnosis  of  typhoid  fever  extending  over  seven  years,  it  may 
be  said  that  this  method  of  diagnosis  is  simple  and  easy  of  performance 
in  the  laboratory  by  an  expert  bacteriologist,  but  it  is  not  to  be  recom- 
mended for  routine  employment  by  practising  physicians  as  a  clinical 
test  unless  they  have  had  experience;  that  with  the  modifications  as 
now  employed,  and  due  regard  to  the  avoidance  of  all  possible  sources 
of  error,  it  is  as  reliable  a  method  as  any  other  bacteriological  test  at 
present  in  use;  and  that  as  such  the  Gruber-Widal  test  is  an  indis- 
pensable, though  not  absolutely  infallible,  aid  to  the  clinical  diagnosis 
of  irregular  or  slightly  marked  typhoid  fever. 

Isolation  of  Typhoid  Bacilli  from  Suspected  Feces,  Urine,  Blood,  Water, 
etc. — In  the  bacteriological  study  of  typhoid  infection  for  diagnostic 
and  other  purposes,  attempts  have  been  made  to  isolate  the  specific 
bacilli  from  the  blood,  rose  spots,  sweat,  urine,  feces,  and  by  spleen 
puncture.  Although  the  results  obtained  by  puncture  of  the  spleen 
have  been  encouraging  and  have  thrown  light  upon  the  distribution 
of  the  organism  in  the  body  during  life,  yet  as  a  regular  means  of  diag- 
nosis it  is  to  be  discouraged,  on  account  of  the  possible  danger  to  the 
patient.  The  results  of  the  examination  of  the  blood  and  rose  spots 
of  typhoid  patients  have  in  the  main  proved  unsatisfactory,  though 
the  investigations  of  some  of  the  later  observers  have  given  a  large 
percentage  of  positive  results  from  the  blood.  The  examination  of  the 
urine  and  feces  of  typhoid  patients  has  more  often  given  positive 
results  than  the  blood,  and  these  positive  results  have  become  more 
frequent  and  satisfactory  as  the  methods  for  differentiating  the  bacillus 
typhosus  have  grown  more  exact  and  refined. 

Several  media  recently  devised  for  the  isolation  and  identification  of 
the  typhoid  bacillus  are  much  better  than  any  of  those  formerly  used. 
These  are  the  Hiss,  Capaldi,  Conradi,  Drigalski,  and  Eisner  media. 
In  the  hands  of  trained  bacteriologists  they  give  satisfactory  results. 
The  first  three  suffice  for  all  ordinary  purposes. 


THK  T}  I'lIOID  BACILLUS  279 

THE  Hiss  MEDIA:  THEIR  COMPOSITION  AND  PREPARATION/ — Two 
media  are  used:  one  for  the  isolation  of  the  typhoid  bacillus  by  plate 
culture,  and  one  for  the  differentiation  of  the  typhoid  bacillus  from  all 
other  forms  in  pure  culture  in  tubes. 

The  platiny  medium  is  composed  of  10  grams  of  agar,  25  grams  of 
gelatin,  5  grams  of  sodium  chloride,  5  grams  of  Liebig's  beef  extract, 
10  grams  of  glucose,  and  1000  c.c.  of  water.  When  the  agar  is  thor- 
oughly melted  the  gelatin  is  added  and  completely  dissolved  by  a  few 
minutes'  boiling.  The  medium  is  then  titrated,  to  determine  its  reac- 
tion, phenolphthalein  being  used  as  the  indicator.  The  requisite 
amount  of  normal  hydrochloric  acid  or  sodium  hydrate  solution  is 
added  to  bring  it  to  the  desired  reaction — i.  e.,  a  reaction  indicating 
2  per  cent,  of  normal  acid.  To  clear  the  medium  add  one  or  two  eggs, 
well  beaten  in  25  c.c.  of  water,  boil  for  forty-five  minutes,  and  filter 
through  a  thin  filter  of  absorbent  cot- 
ton. Add  the  glucose,  after  clearing. 
The  reaction  of  the  medium  is  most 
important  ;  it  should  never  contain 
less  than  2  per  cent,  of  normal  acid. 

The  tube  medium  contains  agar,  5     fit 
grains;    gelatin,    80     grams;    sodium 
chloride,  5    grams  ;    meat   extract,    5  t ! 
grams,  and  glucose,  10  grams  to  the 
litre  of  water,  and  reacts  1.5  per  cent, 
acid    by    the    indicator.     The    mode 
of  preparation  is  the  same  as  for  the 
plate  medium,  care  being  taken  always 
to  add  the   gelatin  after  the   agar  is 

thoroughlv    melted,  SO   as  not    to    alter       HI»«  plate  media:  Small  light  colony  «) 
, !  .      .   c      ' , .       ,   ,  !  ,  is  composed  of  tvphoid  bacilli ;  large  colony 

this  ingredient  by  prolonged  exposure   (c)  of  colon  baciiii.   (From  Hiss.) 

to  high  temperature.     The  glucose  is 

added    after  clearing.     The   medium  must  contain   1.5  per  cent,  of 

normal  acid. 

Growth  of  the  Colonies. — The  growth  of  the  typhoid  bacilli  in  plates 
made  from  the  medium  as  above  described  gives  rise  to  small  colonies 
with  irregular  outgrowth  and  fringing  threads  (Fig.  94).  The  colon 
colonies,  on  the  other  hand,  are  much  larger,  and,  as  a  rule,  are 
darker  in  color  and  do  not  form  threads.  The  growth  of  the  typhoid 
bacilli  in  tubes  produces  uniform  clouding  at  37°  C.  within  eighteen 
hours.  The  colon  cultures  do  not  give  the  uniform  clouding,  and 
present  several  appearances,  probably  dependent  upon  differences  in 
the  degree  of  their  motility  and  gas-producing  properties  in  media. 
Some  of  the  varieties  of  the  colon  bacillus  grow  only  locally  where  they 
were  inoculated  by  the  platinum  needle.  Others  grow  diffusely  through 
the  medium,  but  owing  to  the  production  of  gas  and  the  passage  of 

1  This  description  is  taken  from  an  article  by  Dr.  Philip  Hanson  Hiss,  Jr.,  "On  a  Method  of  Iso- 
lating and  Identifying  Bacillus  Typhosus  and  Members  of  the  Colon  Group  in  Semisolid  Culture 
Media,"  published  in  the  Journal  of  Experimental  Medicine,  1897,  vol.  ii.,  No.  6. 


280  BACTERIA  PATHOGENIC  TO  MAN 

gas-bubbles  through  the  medium,  clear  streaks  ramify  through  the 
otherwise  diffusely  cloudy  tube  contents.  This  characteristic  appear- 
ance is  not  produced  when  the  medium  is  incorrect  in  reaction  or  in 
consistency.  With  untried  media  it  is  always  well  to  insert  a  platinum 
wire  into  the  tube  contents  and  stir  them  about;  if  any  gas  is  liberated 
the  culture  is  not  one  of  the  typhoid  bacillus  and  the  medium  is  not 
correct. 

Method  of  Making  the  Test. — The  usual  method  of  making  the  test 
is  to  take  enough  of  the  specimen  of  feces  or  urine — i.  e.,  from  one  to 
several  loops — and  transfer  it  to  a  tube  containing  broth.  From  this 
emulsion  in  broth  five  or  six  plates  are  generally  made  by  transferring 
one  to  five  loops  of  the  emulsion  to  tubes  containing  the  melted  plate 
medium,  and  then  pouring  the  contents  of  these  tubes  into  Petri  dishes. 
These  dishes  are  placed  in  the  incubator  at  37°  C.  and  allowed  to  remain 
for  eighteen  to  twenty-four  hours,  when  they  may  be  examined.  If 
typical  thread-forming  colonies  are  found  the  tube  medium  is  inocu- 
lated from  them,  and  the  growth  in  the  tubes  allowed  to  develop  for 
about  eighteen  hours  at  37°  C.  If  these  tubes  then  present  the  charac- 
teristic clouding,  experience  indicates  that  the  diagnosis  of  typhoid 
may  be  safely  made,  for  the  typhoid  bacillus  alone,  of  all  the  organisms 
investigated,  has  displayed  the  power  of  giving  rise  both  to  the  thread- 
forming  colonies  in  the  plating  medium  and  the  uniform  clouding  in 
the  tube  medium  when  exposed  to  a  temperature  of  37°  C.  The  organ- 
isms isolated  in  this  manner  have  been  subjected  to  the  usual  tests  for 
the  recognition  of  the  bacillus  typhosus,  and  have  always  corresponded 
in  all  their  reactions  to  those  given  by  the  typical  typhoid  bacillus. 

THE  CAPALDI  PLATE  MEDIUM. — In  his  original  paper,  Capaldi 
gives  the  following  recipe: 

Aquadest. 1000 

Gelatin 20 

Mannite  (grape-sugar)         .         .         .         .         .         .  10 

Sodium  chloride 5 

Potassium  chloride     .......  5 

Boil,  filter,  add  2  per  cent,  agar  and  10  c.c.  of  normal  sodic  hydrate  solution  ;  boil, 
filter,  and  sterilize. 

In  making  up  the  medium  for  work  the  only  variation  was  that  in 
the  original  recipe  the  agar  was  added  when  the  gelatin  was  put  in,  and 
the  gelatin  was  added  after  the  first  filtration. 

The  Capaldi  medium  is  usually  employed  for  surface  cultures,  but 
can  be  inoculated  while  melted  in  the  tubes.  Plates  may  be  made 
beforehand,  so  that  they  are  ready  for  use  when  the  specimen  comes 
in.  As  these  plates  are  to  be  kept  at  37°  C.,  the  difficulties  in  regard 
to  temperature  are  avoided;  but,  unlike  the  Eisner  plates,  other  organ- 
isms besides  the  colon  and  typhoid  develop  and  may  cause  some  con- 
fusion. In  making  the  plates  one  or  two  are  inoculated  by  gently  carry- 
ing across  their  surface  a  platinum  loop  of  feces  or  urine.  Others  are 


THE  TYPHOID  BACILLUS 


281 


then  inoculated  with  a  loop  of  urine  or  much  diluted  feces.  In  this 
way  we  are  apt  to  have  some  plates  with  just  the  right  amount  of 
colonies. 

Appearance  of  the  Colonies. — Capaldi  thus  describes  the  differentia- 
tion :  Typhoid — Small,  glistening,  transparent,  almost  colorless  colonies 
(by  reflected  light,  blue).  Colon — Large,  milky  colonies  (reflected 
light,  brown). 

In  using  the  medium  it  was  found  that  even  in  a  pure  plate  of  typhoid 
the  colonies  vary  much  in  size  and  appearance,  while  different  typhoids 
show  individual  differences  in  growth.  In  general,  a  medium-sized, 
gray-white  colony,  with  a  few  refractive  granules,  is  the  typhoid. 
However,  it  is  often  transparent,  without  the  refractive  granules; 
sometimes  with  a  nuclear  centre,  and  sometimes  of  equal  consistency 
throughout.  Streptococci  simulate  typhoid,  but  a  high-power  lens  will 
show  the  coccus. 

Colon  colonies  are  usually  much  larger  than  the  typhoid — a  decided 
brown  color,  very  large,  refractive  granules,  and  in  general  quite  dif- 
ferent in  appearance  (Fig.  95). 

FIG.  95 


Colonies  of  colon  bacilli  on  Capaldi  medium  slightly  magnified.    Typhoid  colonies  of  same 
size  usually  have  no  dark  granules. 

The  best  way  to  work  with  the  Capaldi  medium  is  to  make  several 
plates  with  different  typhoid  cultures,  observe  carefully  all  the  varia- 
tions in  the  colonies,  and  bear  these  in  mind  when  working  with  the 
mixed  plates.  After  these  precautions  have  been  taken  the  medium 
will  be  found  very  satisfactory.  The  colonies,  as  a  rule,  appear  char- 
acteristically in  twelve  to  eighteen  hours,  and  thus  give  a  quick  method 
of  diagnosis. 

We  found  that  the  two  media  (Capaldi  and  Hiss)  work  excellently 
together,  as  one  is  an  aid  to  the  other.  When  many  colonies  of  the 
typhoid  bacilli  were  present  the  points  of  differentiation  were  usually 


282  BACTERIA  PATHOGENIC  TO  MAN 

easily  seen  upon  both  media,  and  the  two  together  made  diagnosis 
almost  certain.  The  bacilli  from  the  suspected  typhoid  colonies  can 
be  quickly  tested,  sufficiently  for  practical  purposes,  on  the  Hiss  tube 
medium,  and  by  the  reaction  between  the  bacilli  and  the  serum  from 
an  immunized  horse. 

TYPHOID  MEDIUM  OF  VON  DRIGALSKI  AND  CONRADI. — These  authors 
modified  lactose  litmus  agar  by  adding  to  it  nutrose  and  crystal  violet 
and  by  using  3  per  cent,  of  agar  instead  of  2  per  cent.  The  crystal 
violet  strongly  inhibits  the  growth  of  many  other  bacteria,  especially 
cocci,  which  would  also  color  the  medium  red;  the  3  per  cent,  agai 
makes  the  diffusion  of  the  acid  which  is  formed  more  difficult. 

Three  pounds  of  chopped  beef  are  allowed  to  stand  twenty-four 
hours  with  2  litres  of  water.  The  meat  infusion  is  boiled  one  hour 
and  filtered.  20  grams  Witte's  peptone,  20  grams  nutrose,  and  10 
grams  of  salt  are  then  added,  and  the  mixture  boiled  another  hour. 
After  filtration  and  the  addition  of  60  grams  agar  the  mixture  is  boiled 
for  three  hours,  alkalized  and  filtered.  In  the  mean  time  300  c.c. 
litmus  solution  (Kahlbaum)  are  boiled  for  fifteen  minutes  with  30 
grams  lactose.  Both  solutions  are  then  mixed  and  the  mixture,  which 
is  now  red,  faintly  alkalized  with  10  per  cent,  soda  solution.  To  this 
feebly  alkaline  mixture  4  c.c.  hot  sterile  10  per  cent,  soda  solution 
are  added  and  20  c.c.  of  a  sterile  solution  (0.1:100)  of  crystal  violet 
Hochst  B. 

Plates  are  made  of  this  in  the  usual  way.  The  material  to  be 
examined  (stools  first  diluted  with  ten  volumes  of  0.8  per  cent,  salt 
solution)  is  spread  directly  on  the  surface  of  the  plates,  and  these  then 
allowed  to  stand  slightly  open  for  about  half  an  hour  in  order  that 
they  may  dry  somewhat.  They  are  then  placed  inverted  into  the 
incubator  for  from  sixteen  to  twenty-four  hours.  Typhoid  colonies  are 
small  (1  to  3  mm.),  transparent,  and  blue;  colon  colonies  are  red, 
coarser,  less  transparent,  and  larger.  The  suspected  colonies  can  at 
once  be  tested  for  agglutination  with  a  high  grade  typhoid  serum. 

In  general  this  method  has  withstood  critical  tests  and  it  is  nowadays 
regarded  as  one  of  the  very  best. 

As  to  the  comparative  merits  of  the  four  media,  it  is  probably  safe 
to  say  that  any  one  of  them  will,  in  the  hands  of  one  accustomed  to 
them,  reveal  the  typhoid  bacilli,  except  perhaps  when  they  exist  in 
only  the  most  minute  numbers.  The  Eisner  method  has  the  objec- 
tion that  it  is  very  difficult  to  work  with  in  hot  weather.  The  Hiss 
plate  medium  has  the  objection  that  it  is  a  difficult  medium  to  pre- 
pare. If  the  acidity  is  not  just  right  the  thread  outgrowths  do  not 
appear.  Indeed,  the  only  sure  way  is  to  test  a  new  batch  of  medium 
with  a  pure  culture  and  alter  the  reaction  until  the  culture  grows  cor- 
rectly. A  very  few  varieties  of  the  typhoid  bacillus  do  not  produce 
typical  thread  outgrowths  from  the  colonies.  In  the  Drigalski 
medium  the  typhoid  colonies  are  easily  separated  from  those  of  the 
colon  bacilli,  but  there  are  other  intestinal  bacteria  which  grow  like 
them. 


THE  TYPHOID  BACILLI  S  283 

The  Cupaldi  medium  lias  the  objection  that  some  of  the  typhoid 
and  some  of  the  colon  colonies  frequently  look  much  alike.  If  one, 
however,  will  always  pick  out  the  colonies  which  look  most  like  the 
typhoid,  it  will  usually  turn  out  that  typhoid  bacilli  have  been  obtained 
if  any  were  present.  Personally,  for  general  use,  I  prefer  the  Capaldi 
or  Drigalski  medium  for  the  plate  cultures  and  the  Hiss  tube  medium 
for  identifying  the  bacilli  obtained.  Through  these  media  and  specific 
agglutinating  serum  we  are  now  in  a  position  to  obtain  and  identify 
typhoid  bacilli  from  feces,  urine,  etc.,  within  forty-eight  hours. 

Typhoid  Bacilli  in  Feces. — Recently  numerous  investigations  have 
been  carried  out  to  discover  how  frequently  and  at  what  period  in 
typhoid  fever  bacilli  were  present  in  the  feces  and  urine.  Hiss  some 
time  ago  examined  the  feces  in  43  consecutive  cases,  37  of  which  were 
in  the  febrile  stage  and  6  convalescent.  In  a  number  of  instances 
only  one  stool  was  examined,  but  even  under  these  adverse  conditions 
the  average  of  positive  results  in  the  febrile  stage  was  66.6  per  cent. 
Out  of  26  cases  of  typhoid  fever  examined  in  hospitals,  21  were  in 
the  febrile  stage  and  5  convalescent.  In  the  febrile  cases  in  17  the 
presence  of  typhoid  bacilli,  often  in  great  numbers,  was  demonstrated. 
Thus  in  these  carefully  followed  cases  the  statistics  show  over  80  per 
cent,  of  the  febrile  cases  positive.  The  bacilli  were  isolated  from  these 
cases  as  early  as  the  sixth  day,  and  as  late  as  the  thirtieth  day,  and  in 
a  case  of  relapse  on  the  forty-seventh  day  of  the  disease.  The  con- 
valescent cases  gave  uniformly  negative  results,  the  earliest  examination 
having  been  made  on  the  third  day  after  the  disappearance  of  the  fever. 
The  bacilli  seemed  to  be  more  numerous  in  the  stools  from  about  the 
tenth  or  twelfth  day  on.  These  observations,  with  regard  to  the  appear- 
ance of  the  bacilli  in  the  stools  during  the  febrile  stage  and  their  usually 
quick  disappearance  after  the  defervescence,  have  been  confirmed  by 
others.  The  bacilli  were  isolated  in  several  cases  in  which  no  Widal 
reaction  was  demonstrated.  Between  the  seventh  and  twenty-first  days 
of  the  disease,  experience  seems  to  indicate  that  the  bacilli  may  be 
obtained  from  about  25  per  cent,  of  all  cases  on  the  first  examination 
and  from  about  75  per  cent,  after  repeated  examinations.  In  some 
samples  of  feces  typhoid  bacilli  die  out  within  twenty-four  hours;  in 
others  they  remain  alive  for  days  or  even  weeks.  This  seems  to  depend 
on  the  bacteria  present  in  the  feces  and  upon  its  chemical  character. 
Probably  the  presence  of  typhoid  bacilli  in  some  stools  and  their 
absence  in  others  must  be  explained  largely  by  the  characteristics  of 
the  intestinal  contents.  The  short  life  of  the  typhoid  bacillus  in  many 
specimens  of  feces  suggests  that  stools  be  examined  as  quickly  as  pos- 
sible. In  fact,  unless  the  physician  wishes  to  take  the  trouble  to  have 
the  sample  of  feces  sent  immediately  to  the  laboratory,  it  is  hardly 
worth  while  for  the  bacteriologist  to  take  the  trouble  to  make  the 
test. 

Typhoid  Bacilli  in  the  Urine. — Of  even  more  interest  than  the  pres- 
ence of  the  bacilli  in  feces  is  their  frequent  occurrence  in  great 
numbers  in  the  urine.  The  results  of  the  examinations  of  others  as 


284  BACTERIA  PATHOGENIC  TO  MAN 

well  as  our  own  indicate  that  the  typhoid  bacilli  are  not  apt  to  be 
found  in  the  urine  until  the  beginning  of  the  third  week  of  the  fever, 
and  may  not  appear  until  much  later.  From  this  on  to  convalescence 
they  appear  in  about  25  per  cent,  of  the  cases,  usually  in  pure  culture 
and  in  enormous  numbers.  Of  9  positive  cases  examined  by  Richard- 
son1 2  died  and  7  were  discharged.  At  the  time  of  their  discharge 
their  urine  was  loaded  with  typhoid  bacilli.  We  have  observed  similar 
cases.  In  one  the  bacilli  persisted  for  five  weeks.  Undoubtedly  in 
exceptional  cases  they  persist  for  years.  When  we  think  of  the  chances 
such  cases  have  to  spread  infection  as  they  pass  from-  place  to  place, 
we  begin  to  realize  how  epidemics  can  start  without  apparent  cause. 
The  more  we  investigate  the  persistence  of  bacteria  in  convalescent 
cases  of  disease,  the  more  difficult  the  prevention  of  their  dissemina- 
tion is  seen  to  be.  The  disinfection  of  the  urine  should  always  be 
looked  after  in  typhoid  fever,  and  convalescents  should  not  be  allowed 
to  go  to  places  where  contamination  of  the  water  supply  is  possible r 
without  at  least  warning  them  of  the  necessity  of  great  care  in  disin- 
fecting their  urine  and  feces  for  some  weeks.  Richardson  made  the 
interesting  discovery  that  after  washing  out  the  bladder  with  a  very 
weak  solution  of  bichloride  of  mercury  the  typhoid  bacilli  no  longer 
appeared  in  the  urine. 

Paratyphoid. — A  few  of  the  cases  of  "  typhoid  "  heretofore  described 
as  giving  no  Gruber-Widal  reaction  were  undoubtedly  due  to  the  para- 
typhoid bacilli.  As  has  been  already  stated,  this  is  the  name  by  which 
we  now,  in  conformity  with  Schottmuller,  designate  a  bacillrs  which 
stands  about  midway  between  B.  typhosus  and  B.  coli.  It  has  been 
found  necessary  to  distinguish  two  varieties,  type  A  and  type  B,  which 
differ  also  in  their  agglutinating  property.  It  remains  to  be  seen  whether 
we  shall  have  to  differentiate  any  additional  types.  There  are  no  certain 
distinguishing  features  to  separate  the  clinical  pictures  of  abdominal 
typhoid  and  paratyphoid.  Many  cases  of  paratyphoid  present  all  the 
classical  symptoms  of  typhoid.  According  to  Conradi,  von  Drigalski, 
and  Jiirgens  the  fever  curve  of  paratyphoid  is  characterized  by  a  fairly 
sudden  rise,  an  irregular  course  of  the  temperature  with  almost  always 
an  absence  of  the  continua.  Besides  this,  the  disease  has  a  better 
prognosis  and  a  slow  convalescence.  According  to  other  authors, 
enlargement  of  the  spleen  is  quite  often  absent  (de  Feyfer  and  Kayser 
missed  it  in  42  per  cent,  of  the  cases),  whereas  an  involvement  of  the 
upper  portions  of  the  intestinal  tract  (gastric  fever!)  is  more  common. 
Further  than  this,  it  is  unwise  to  lay  much  stress  on  peculiarities  in  the 
course  of  the  disease,  for  we  know  that  true  typhoid  runs  a  variable 
course.  We  have  only  to  think  of  the  vast  difference  between  a  mild 
or  abortive  typhoid  and  a  fully  developed  or,  better  still,  a  complicated 
case.  It  will  almost  always  be  impossible  to  separate  a  case  of  true 
typhoid  from  a  paratyphoid  by  the  symptoms  alone.  At  the  most, 
during  an  epidemic  the  general  course  of  the  disease,  when  it  agrees 

1  Journal  of  Experimental  Medicine,  May,  1898. 


T11K  TYPHOID  BACILLUS  285 

with  the  above  points,  may  cause  one  to  suspect  paratyphoid.  Schott- 
miiller  and  Kurth,  from  a  total  of  180  cases  which  had  been  looked 
upon  as  typhoid,  were  able  in  12  cases  to  isolate  a  paratyphoid 
bacillus. 

llfmermaim  observed  a  whole  epidemic  in  which  typhoid-like  bac- 
teria, which  he  regarded  as  the  cause  of  the  disease,  were  found  in 
the  blood.  The  Gruber-Widal  reaction  1 : 100  was  positive  in  only  42 
per  cent.;  the  newly  found  bacillus  was  always  agglutinated.  Similar 
reports  concerning  an  epidemic  of  14  cases  in  Holland  are  made  by 
de  Feyfer  and  Kayser;  and  Sion  and  Negel  report  one  from  Roumania. 
Formerly  none  of  these  cases  would  have  been  differentiated  from  true 
typhoid. 

Detection  of  Typhoid  Bacilli  in  Water. — There  is  absolutely  no  doubt 
that  the  contamination  of  streams  and  reservoirs  is  a  frequent  cause 
of  the  outbreak  of  epidemics  of  typhoid  fever,  but  the  actual  finding 
and  isolation  of  the  bacilli  is  a  very  rare  occurrence.  This  is  often 
due  to  the  fact  that  the  contamination  has  passed  away  before  the 
bacteriological  examination  is  undertaken,  and  also  to  the  great 
difficulties  met  with  in  detecting  a  few  typhoid  bacilli  when  they  are 
associated  with  large  numbers  of  other  bacteria.  The  greater  the 
amount  of  contamination  entering  the  water,  and  the  shorter  the  time 
which  elapses  between  this  and  the  drinking  of  the  water,  the  greater  is 
the  danger. 

Differential  Diagnosis. — The  typhoid  bacillus  and  the  bacilli  of  the 
colon  group  resemble  each  other  in  many  respects.  It  is  necessary  to 
remember  that  there  are  many  varieties  of  bacilli  differing  in  both  cultural 
and  agglutination  reactions  which  are  grouped  under  the  general  name 
of  the  colon  bacillus.  By  comparing  what  has  been  said  of  the 
bacillus  coli  and  the  bacillus  typhosus  it  will  be  seen  that  while  certain 
varieties  of  each  simulate  each  other  in  many  respects,  the  character- 
istic varieties  of  each  still  possess  individual  characteristics  by  which 
they  may  be  readily  differentiated: 

1.  The  motility  of  the  B.  coli  is,  as  a  rule,  much  less  marked  than 
that  of  the  B.  typhosus.    Tne  cobn  bacillus  is  also  shorter,  thicker,  and 
has  fewer  flagella. 

2.  In  gelatin  the  colonies  of  the  colon  bacillus  develop  more  rapidly 
and  luxuriantly  than  those  of  the  typhoid  bacillus. 

3.  On  potato  the  growth  of  the  colon  bacillus  is  usually  rapid,  luxuri- 
ant, and  visible,  though  not  invariably  so;  while  that  of  the  typhoid 
bacillus  is  ordinarily  invisible. 

4.  The  characteristic  colon  bacillus  coagulates  milk  in  from  thirty- 
six  to  forty-eight  hours  in  the  incubator,   with  acid  reaction.     The 
typhoid  bacillus  does  not  cause  coagulation. 

5.  The  colon  bacillus  is  conspicuous  for  its  power  of  causing  fer- 
mentation, with  the  production  of  gas  in  media  containing  glucose. 
The  typhoid  bacillus  never  does  this. 

6.  In  nutrient  agar  or  gelatin  containing  lactose  and  litmus  tincture, 
and  of  a  slightly  alkaline  reaction,  the  color  of  the  colonies  of  the  colon 


286  BACTERIA  PATHOGENIC  TO  MAN 

bacillus  is  pink,  and  the  surrounding  medium  becomes  red;  while  the 
colonies  of  the  typhoid  bacillus  are  blue,  and  there  is  little  or  no  red- 
dening of  the  surrounding  medium.  The  same  points  hold  true  on 
the  Drigalski-Conradi  medium. 

7.  The  colon  bacillus  possesses  the  property  of  producing  indol  in 
cultures   of  bouillon   or  peptone;  the    characteristic   typhoid   bacillus 
does  not  produce  indol  in  these  solutions. 

8.  The  colon  bacillus  rarely  produces  thread  outgrowths  in  properly 
prepared  Hiss  plate  medium.     The  typhoid  bacillus  produces  thread 
outgrowths  and  smaller  colonies  in  this  medium.     In  the  Hiss  tube 
medium  the  colon  bacillus  produces  either  a  growth  limited  to  the 
area  inoculated  or  a  diffuse  growth  streaked  with  clear  lines  and  spaces. 
The  typhoid  bacillus  produces  a  diffuse  growth,  evenly  clouding  the 
entire  medium. 

9.  On  the  Capaldi  medium  the  colon  colonies  are  more  granular 
and  darker  than  those  of  the  typhoid  bacilli. 

10.  Finally,   on  testing   the   bacilli   in   the  hanging  drop  with  the 
serum  of  animals  immunized  to  the  typhoid  bacillus,  the  typhoid  bacilli 
become  agglutinated  in  high  dilutions  of  the  serum,  while  the  colon 
bacilli  do  not. 

None  of  these  tests  alone  can  be  depended  upon  for  making  a  differ- 
ential diagnosis  of  the  colon  bacillus  from  the  typhoid  bacillus  or  other 
similar  bacilli. 

Unfortunately,  also,  in  most  of  these  characteristics  certain  degrees 
of  variation  may  often  be  observed  and  these  may  lead  to  confusion. 
For  instance,  the  morphology  may  vary  considerably,  at  times  even 
when  grown  on  the  same  culture  media,  and  the  motility  is  not  always 
equally  pronounced;  the  flagella  may  vary;  the  rapidity  of  growth 
may  differ,  especially  between  freshly  made  and  old  cultures;  the 
grape-leaf  appearance  of  the  surface  colonies  on  gelatin,  which  is 
usually  characteristic,  may  vary  with  the  composition  of  the  gelatin, 
at  times  no  typical  colonies  at  all  being  presented;  the  threads  in  the 
Hiss  media  may  be  lacking;  in  rare  instances  the  typhoid  bacillus  pro- 
duces indol;  the  growth  on  potato  is  not  to  be  depended  on,  often 
being  visible  and  not  characteristic;  the  virulence  of  both  the  bacilli 
is  so  little  characteristic  that  it  can  hardly  be  used  for  diagnostic  pur- 
poses; and  finally,  the  serum  test  is  not  to  be  depended  on  unless  the 
agglutinins  in  the  serum  have  been  properly  tested,  for  there  is 
abundant  agglutinin  for  some  of  the  colon  bacilli  in  the  serum  of 
many  untreated  animals.  This  is  less  true  of  rabbits  than  of  horses 
and  of  young  than  older  animals. 

In  spite,  however,  of  these  difficulties  it  is  very  easy  to  sufficiently 
identify  the  typhoid  bacillus  for  all  practical  purposes.  A  bacillus 
which  grows  typically  in  the  Hiss  tube  media,  and  shows  agglutination 
with  a  high  dilution  of  the  serum  of  an  animal  immunized  to  the  typhoid 
bacillus,  is  in  all  probability  the  typhoid  bacillus.  If  this  bacillus 
absorbs  the  specific  typhoid  agglutinins  it  is  undoubtedly  the  typhoid 
bacillus.  The  same  could  probably  be  said  of  a  bacillus  which  grew 


THE  TYPHOID  BACILLUS  287 

characteristically  in  glucose  bouillon  and  nutrient  gelatin,  besides 
showing  the  specific  serum  reaction.  Probably  not  one  time  in  ten 
thousand  would  such  bacilli  prove  on  further  investigation  not  to  be 
typhoid  bacilli.  A  still  further  test  is  to  inoculate  animals  with  several 
doses  of  the  dead  bacilli  whose  identification  is  sought,  and  note  whether 
there  is  produced  a  serum  which  specifically  agglutinates  undoubted 
typhoid  bacilli. 


CHAPTER  XXL 

THE  BACILLUS  OF  TUBERCULOSIS. 

A  KNOWLEDGE  of  phthisis  was  certainly  present  among  men  at  the 
time  from  which  our  earliest  medical  descriptions  come.  For  over 
two  thousand  years  many  of  the  clearest-thinking  physicians  have 
considered  it  a  communicable  disease;  but  it  is  only  within  compara- 
tively recent  times  that  the  infectiousness  of  tuberculosis  has  become 
an  established  fact  in  scientific  medicine.  Villemin,  in  1865,  by  infecting 
a  series  of  animals  through  inoculations  with  tuberculous  tissue,  showed 
that  tuberculosis  might  be  induced,  and  that  such  tissue  carried  the 
exciting  agent  of  the  disease.  Baumgarten  demonstrated,  early  in  1882, 
bacilli  in  tissue  sections  which  are  now  known  to  have  been  the  tubercle 
bacilli.  But  these  investigations  and  those  of  others  at  the  same  time, 
though  paving  the  way  to  a  better  knowledge  of  the  disease,  proved  to 
be  unsatisfactory  and  incomplete.  The  announcement  of  the  discovery 
of  the  tubercle  bacillus  was  made  by  Koch  in  March,  1882.  Along 
with  the  announcement  satisfactory  experimental  evidence  was  pre- 
sented as  to  its  etiological  relation  to  tuberculosis  in  man  and  in  sus- 
ceptible animals,  and  its  principal  biological  characters  were  given. 
An  innumerable  number  of  investigators  now  followed  Koch  into  this 
field,  but  their  observations  served  only  to  confirm  his  discovery. 

Distribution  of  Bacilli. — They  are  found  in  the  sputum  of  persons 
and  animals  suffering  from  pulmonary  or  laryngeal  tuberculosis,  either 
free  or  in  the  interior  of  pus  cells;  in  miliary  tubercles  and  fresh  caseous 
masses  in  the  lungs  and  elsewhere;  in  recent  tuberculous  cavities  in  the 
lungs;  in  tuberculous  glands,  joints,  bones,  mucous  membranes,  and 
skin  affections. 

Morphology. — The  tubercle  bacilli  are  slender,  non-motile  rods  of 
about  0.3/*  in  diameter  by  1.5  to  4//.  in  length.  (Plate  I.,  Figs.  1,  2, 
and  3.)  Commonly  they  occur  singly  or  in  pairs,  and  are  then  usually 
slightly  curved;  frequently  they  are  observed  in  smaller  or  larger  bunches. 
Under  exceptional  conditions  branching  and  club-shaped  forms  are 
observed.  Injected  into  the  brain,  kidney,  and  other  tissues  in  rabbits 
a  growth  frequently  occurs  in  which  forms  similar  to  actinomyces 
develop.  The  tubercle  bacillus  clearly  belongs  among  the  higher  forms 
of  bacteria  and  is  closely  allied  to  actinomycosis.  The  same  is  true  for 
some  of  the  timothy  and  other  acid-fast  bacilli.  In  stained  preparations 
there  are  often  seen  unstained  portions.  From  two  to  six  of  these 
vacuoles  may  sometimes  be  noticed  in  a  single  rod.  In  old  cultures 
irregular  forms  may  develop,  the  rods  being  occasionally  swollen  at 
one  end  or  presenting  lateral  projections.  Here  also  spherical  granules 


PLATE    I. 


FIG.  1. 


FIG.  2. 


Tubercle  bacilli,  in  red. 
Streptobaeilli,  in  blue. 

X  11OO  diameters. 


Tubercle  bacilli,  in  red. 
Tissue,  in  blue. 

X  HOO  diameters 


FIG.  3. 


FIG.  4. 


Leprosy  bacilli  in  nasal  secre- 
tion of  person  suffering  from 
nasal  lesions.  (Hansen.) 


Short  smegma  bacilli. 

Bacilli  in  specimen  are  red, 

rest  of  material  in  blue. 


X  3OO  diameters. 


11OO  diameters. 


THE  BACILLUS  OF  TUBERCULOSIS  289 

appear  which  stain  with  more  difficulty  than  the  rest  of  the  bacillus 
and  also  retain  the  stain  with  greater  tenacity.  The  bacilli,  however, 
containing  these  bodies  are  not  appreciably  more  resistant  than  those 
not  having  them;  although,  therefore,  these  bodies  have  some  of  the 
characteristics  of  a  spore,  they  lack  the  quality  of  resistance  to  dele- 
terious influences  and  cannot  be  considered  true  spores. 

The  bacilli  have  a  thin  capsule,  shown  in  one  way  by  the  fact  that 
they  appear  thicker  when  stained  with  fuchsin  than  with  methylene 
blue.     The  capsule  is  believed  to  contain  the 
greater  portion  of  the  wax-like  substance  pecu-  Fl°- 96 

liar  to  the  bacillus. 

Staining  Peculiarities.  —  These  are  very  im- 
portant, for  by  them  its  differentiation  and 
recognition  in  microscopic  preparations  of 
sputum,  etc.,  are  rendered  possible.  Owing  to 
the  waxy  substance  in  its  envelope  it  does  not 
readily  take  up  the  ordinary  aniline  colors,  but 
when  once  stained  it  is  very  difficult  to  decol- 
orize, even  by  the  use  of  strong  acids.  The 
more  recently  formed  bacilli  are  much  more 
easilv  stained  and  decolorized  than  the  older 

f  TM     i-   i       i      •       i  ,110          •    •  Branched  forms.    (From 

forms.     Ehrlich  devised  a  method  of  staining  c.  F.  Craig.) 

which  proved  to  be  satisfactory — viz.,  the  use  of 

a  solution  of  an  aniline  color — fuchsin  or  methyl  violet — in  a  saturated 
aqueous  solution  of  aniline  oil  and  decolorization  of  other  bacteria  with 
a  solution  of  a  mineral  acid,  to  be  followed  by  a  contrast  stain,  such  as 
methylene  blue.  (Plate  I.,  Figs.  1  and  2.)  Various  modifications  of 
Ehrlich's  method  are  now  commonly  used.  The  tubercle  bacilli  can  be 
demonstrated  also  by  Gram's  method  of  staining. 

Biology. — The  bacillus  tuberculosis  is  a  parasitic,  aerobic,  non-motile 
bacillus,  and  grows  only  at  a  temperature  of  about  37°  C.,  limits  30° 
to  42°  C.  It  does  not  form  true  spores. 

RESISTANCE. — The  bacilli,  possibly  on  account  of  the  nature  of  their 
capsule,  have  a  somewhat  greater  resisting  power  than  most  other 
pathogenic  bacteria,  since  frequently  the  bacilli  resist  desiccation  at 
the  ordinary  temperatures  for  months;  most  bacilli  die,  however,  soon 
after  drying.  Upon  serum  cultures  the  bacilli  seldom  live  longer  than 
six  to  eight  months.  They  frequently  retain  their  vitality  for  several 
weeks  in  putrefying  material,  such  as  sputum.  Cold  has  little  effect 
upon  them.  When  dry  the  more  resistant  organisms  stand  dry  heat 
at  100°  C.  for  hours;  but  when  moist,  as  in  milk,  they  are  quickly 
killed — viz.,  at  55°  C.  in  four  hours,  at  60°  C.  in  thirty  minutes,  at 
65°  C.  in  fifteen  minutes,  at  70°  C.  in  ten  minutes,  at  80°  C.  in  five 
minutes,  and  at  95°  C.  in  one  minute.  One  reason  why  in  some 
experiments  they  appear  to  withstand  high  temperatures  is,  as  pointed 
out  by  Theobald  Smith,  that  when  heated  in  a  test-tube  in  the  usual 
way  the  cream  which  rises  on  heating  is  exposed  on  its  surface  to  a 
lower  temperature  than  the  rest  of  the  milk,  and  as  this  contains  the 

19 


290  BACTERIA  PATHOGENIC  TO  MAN 

greatest  percentage  of  the  bacteria  some  of  them  are  exposed  to  less 
heat  than  those  in  the  rest  of  the  fluid  receive. 

The  resisting  power  of  this  bacillus  to  chemical  disinfectants,  drying, 
and  light  is  considerable,  but  not  as  great  as  it  is  apt  to  appear,  for, 
as  in  sputum,  the  bacillus  is  usually  protected  by  mucus  or  cell  pro- 
toplasm from  penetration  by  the  germicidal  agent.  It  is  not  always 
destroyed  by  the  gastric  juice  in  the  stomach,  as  is  shown  by  successful 
infection  experiments  in  susceptible  animals  by  feeding  them  with 
tubercle  bacilli.  They  are  destroyed  in  sputum  in  six  hours  or  less 
by  the  addition  of  an  equal  quantity  of  a  5  per  cent,  solution  of  carbolic 
acid.  Bichloride  of  mercury  is  less  suitable  for  the  disinfection  of 
sputum.  lodoform  has  no  effect  upon  cultures  until  5  per  cent,  is 
added.  The  fumes  from  four  pounds  of  burning  sulphur  to  each 
1000  cubic  feet  of  air  space  will  kill  tubercle  bacilli  in  eight  hours  when 
fully  exposed  to  the  action  of  the  gas,  providing  that  they  are  moist, 
or  that  abundant  moisture  is  present  in  the  air. 

Formaldehyde  gas  is  quicker  in  its  action,  but  not  much  more  efficient. 
Ten  ounces  of  formalin  should  be  employed  for  each  1000  cubic  feet 
of  air  space. 

The  tubercle  bacillus  in  sputum  when  exposed  to  direct  sunlight  is 
killed  in  from  a  few  minutes  to  several  hours,  according  to  the  thickness 
of  the  layer  and  the  season  of  the  year;  it  is  also  usually  destroyed  by 
diffuse  daylight  in  from  five  to  ten  days  when  placed  near  a  window. 
Protected  in  cloth  the  bacilli  survive  exposure  to  light  for  longer  periods. 
This  fact  is  worthy  of  note,  as  it  has  an  important  hygienic  bearing. 
Thus,  tuberculous  sputum  expectorated  upon  sidewalks,  etc.,  being 
often  exposed  to  the  action  of  direct  sunlight,  will  in  many  cases,  especially 
in  summer,  be  disinfected  by  the  time  it  is  in  a  condition  to  be  carried 
into  the  air  as  dust.  For  this  and  other  more  important  hygienic  rea- 
sons, consumptive  patients  should  occupy  light,  sunny  rooms  and  live 
as  much  as  possible  in  the  open  air. 

Dried  sputum  in  places  protected  from  abundant  light  has  occasionally 
been  found  to  contain  virulent  tubercle  bacilli  for  as  long  as  ten  months. 
For  a  year  at  least  it  should  be  considered  dangerous.  The  Roentgen 
rays  have  a  deleterious  effect  on  tubercle  bacilli  in  cultures,  but 
practically  none  upon  those  in  tissues. 

The  tubercle  bacillus  is  a  strict  parasite — that  is  to  say,  its  biological 
characters  are  such  that  it  could  scarcely  find  natural  conditions  outside 
of  the  bodies  of  living  animals  favorable  for  its  multiplication.  Under 
exceptional  conditions,  such  as  in  freshly  expectorated  sputum,  tubercle 
bacilli  may  increase  for  a  limited  time. 

Cultivation  of  the  Tubercle  Bacillus. — On  account  of  their  slow 
growth  and  the  special  conditions  which  they  require,  tubercle  bacilli 
cannot  be  grown  in  pure  culture  by  the  usual  plate  method  on  the 
ordinary  culture  media.  Koch  first  succeeded  in  cultivating  and 
isolating  this  bacillus  on  coagulated  blood  serum,  which  he  inoculated 
by  carefully  rubbing  the  surface  with  sections  of  tuberculous  tissue  and 
then  leav  ng  the  culture,  protected  from  evaporation,  for  several  weeks 


THE  BACILLUS  OF  TUBERCULOSIS  291 

in  the  incubator.  Cultures  are  more  readily  obtained  from  human 
than  from  bovine  bacilli. 

GROWTH  ON  COAGULATED  BLOOD  SERUM  OR  EGG. — On  these  media, 
one  of  wh  ch  is  regularly  used  to  obtain  the  first  culture,  the  growth 
first  becomes  visible  at  the  end  of  ten  to  .twenty-one  days  at  37°  C., 
and  at  the  end  of  three  to  four  weeks  a  distinct  and  characteristic 
development  has  occurred  Small,  grayish-white  points  and  scales  first 
appear  on  the  surface  of  the  medium.  As  development  progresses  there 
is  formed  an  irregular,  membranous-looking  layer.  When  a  tiny  piece 
of  this  is  removed,  placed  on  a  cover-glass  without  rubbing,  stained, 
and  then  observed  under  the  microscope,  the  surface  growth  presents 
a  characteristic  appearance,  the  bacilli  being  arranged  in  parallel  rows 
of  variously  curved  figures. 

Owing  to  the  greater  facility  of  preparing  and  sterilizing  glycerin  agar, 
and  the  more  rapid  and  abundant  growth  of  the  bacilli,  which  have 
become  accustomed  to  growth  outside  the  body  on  this  medium,  it  is 
now  usually  employed  in  preference  to  blood  serum  for  continuing  to 
produce  later  cultures.  The  development  at  the  end  of  fourteen  to 
twenty-one  days  is  more  abundant  than  upon  blood  serum  after  several 
weeks.  When  numerous  bacilli  have  been  distributed  over  the  surface 
of  the  culture  medium,  a  rather  uniform,  thick,  white  layer,  which 
subsequently  acquires  a  slightly  yellowish  tint,  is  developed;  when  the 
bacilli  sown  are  few  in  number,  or  are  associated  in  scattered  groups, 
separate  colonies  are  developed,  which  acquire  considerable  thickness 
and  have  more  or  less  irregular  outlines. 

GROWTH  ON  NUTRIENT  VEAL  OR  BEEF  BROTH  CONTAINING  5  PER 
CENT.  OF  GLYCERIN. — This  is  of  importance,  because  in  this  way  tuber- 

Fio.  97 


Growth  of  tubercle  bacilli  upon  glycerin  bouillon.    (Kolle  and  Wassermann.) 

culin  is  produced.  On  these  media  the  tubercle  bacillus  also  grows 
readily  if  a  very  fresh  thin  film  of  growth  from  the  glycerin  agar  is 
floated  on  the  surface.  The  latter  of  these  media  is  used  for  the  devel- 
opment of  tuberculin.  The  small  piece  of  pellicle  removed  from  the 
previous  culture  continues  to  enlarge  while  it  floats  on  the  surface  of 
the  liquid,  and  in  the  course  of  three  to  six  weeks  covers  it  wholly  as 


292  BACTERIA  PATHOGENIC  TO  MAN 

a  single  film,  which  on  agitation  is  easily  broken  up  and  then  settles 
on  the  bottom  of  the  flask,  where  it  ceases  to  develop  further.  The 
liquid  remains  clear.  A  practical  point  of  importance,  if  a  quick  growth 
is  desired,  is  to  remove  for  the  new  cultures  a  portion  of  the  pellicle 
of  a  growing  bouillon  culture,  which  is  very  thin  and  actively  increasing. 

Obtaining  of  Pure  Cultures  of  the  Tubercle  Bacillus  from  Sputum,  Infected 
Tissue  and  Other  Materials. — On  account  of  the  time  required  and  the 
difficulties  to  be  overcome  this  is  never  desirable  except  when  careful 
investigations  of  importance  are  to  be  undertaken.  The  chief  point  of 
present  interest  is  the  dissemination  of  bovine  bacilli  in  man.  The 
discovery  by  Theobald  Smith  of  the  greater  acid  production  of  the 
human  type  of  tubercle  bacilli  in  glycerin  bouillon  over  the  bovine 
bacilli  has  made  it  a  matter  of  added  importance  to  test  as  many  bacilli 
as  possible  for  their  biochemical  characteristics.  Pure  cultures  .can  be 
obtained  directly  from  tuberculous  material  when  mixed  infection  is 
not  present,  and  a  suitable  dog  serum  or  egg  culture  medium  is  at  hand ; 
but  as  it  is  so  difficult  to  get  material  free  from  other  bacteria  which  grow 
much  more  rapidly  and  take  possession  of  the  medium  before  the 
tubercle  bacillus  has  had  time  to  form  visible  colonies,  it  is  usually 
necessary  first  to  inoculate  a  guinea-pig,  both  subcutaneously  and 
intraperitoneally,  and  then  obtain  cultures  from  the  animal  as  soon  as 
the  tubercle  infection  has  fully  developed.  From  acute  tuberculosis 
in  man  in  other  regions  than  the  lungs  direct  cultures  on  blood  serum 
or  egg  may  be  made  with  some  hope  of  success.  Under  the  best  con- 
ditions great  care  and  patience  are  necessary  if  successful  results  are 
to  be  obtained. 

Animals  inoculated  usually  die  at  the  end  of  three  weeks  to  four 
months.  It  is  better,  however,  not  to  wait  until  death  of  the  animals, 
but  at  the  end  of  three  weeks  to  kill  a  guinea-pig,  which  by  its  enlarged 
glands  shows  evidence  of  tuberculosis,  and  to  remove,  with  the  greatest 
care  as  to  cleanliness,  one  or  more  nodules  from  the  lungs,  spleen,  or 
lymphatic  glands.  Animals  which  develop  tuberculosis  acutely  are  apt 
to  have  abundant  tubercle  bacilli  and  give  successful  cultures,  while  the 
chronic  cases  usually  have  few  bacilli  and  frequently  give  unsuccessful 
cultures.  The  animals  after  being  killed  are  placed  in  trays,  and  after 
washing  with  a  5  per  cent,  solution  of  carbolic  acid,  immediately  autop- 
sied.  The  skin  over  the  anterior  portion  of  the  body  having  been 
carefully  turned  back,  an  opening  is  cut  with  a  fresh  set  of  sterile  instru- 
ments into  the  thoracic  or  abdominal  cavity;  then  with  a  sterile  forceps 
the  lymph-gland,  portion  of  spleen  or  other  part  which  it  is  desired  to 
examine  is  removed  to  a  sterile  covered  beaker.  This  tissue,  if  suitable, 
is  sliced  in  thin  sections  and  conveyed  directly  to  the  surface  of  the 
solid  culture  medium.  It  is  allowed  to  remain  in  contact  with  the 
moist  serum  or  egg  at  37°  C.  for  three  to  ten  days,  and  then  by  means 
of  a  platinum  wire  gently  rubbed  and  drawn  over  the  surface  of  the 
media.  The  tubes  are  then  replaced  in  the  incubator  for  ten  days  to 
three  weeks,  when  a  visible  culture  should  be  obtained.  Owing  to  the 
liability  of  the  blood  serum  to  become  too  dry  for  the  development  of 


TIU-:  itAciu.rs  ///••  Ti'H/-:iu.'i-Losis  293 

the  bacillus,  it  is  necessary  to  keep  the  culture  moist  by  sealing  the 
end  in  some  way.  Theobald  Smith,  who  has  had  a  very  large  experience 
in  growing  the  tubercle  bacillus,  gives  the  following  details  as  to  his 
method: 

"Throughout  the  work  solidified  dog's  serum  was  used  as  being  the 
best  medium.  The  dog  was  bled  under  chloroform  and  the  blood 
drawn  from  a  femoral  artery,  under  aseptic  conditions,  through  sterile 
tubes  directly  into  sterile  flasks.  The  serum  was  drawn  from  the  clot 
with  sterile  pipettes,  and  either  distributed  at  once  into  tubes  or  else 
stored  with  0.25  to  0.3  per  cent,  chloroform  added.  The  temperature 
required  to  produce  a  sufficiently  firm  and  yet  not  too  hard  and  dry 
serum  is,  for  the  dog,  75°  to  76°  C.  The  tubes  containing  the  serum 
were  set  in  a  thermostat,  into  which  a  dish  of  water  was  placed,  to 
forestall  any  abstraction  of  moisture  from  the  serum.  About  three 
hours  suffice  for  the  coagulation.  This  procedure  dispenses  with  all 
sterilization,  excepting  that  going  on  during  the  coagulation  of  the 
serum.  It  prevents  the  gradual  formation  of  membranes  of  salts,  which, 
remaining  on  the  surface  during  coagulation,  form  a  film  unsuited  for 
bacteria.  Tubes  of  coagulated  serum  should  be  kept  in  a  cold,  closed 
space,  where  the  opportunities  for  evaporation  are  slight.  They  should 
always  be  kept  inclined. 

"The  ordinary  cotton-plugged  test-tubes  I  do  not  use,  because  of 
the  rapid  drying  out  permitted  by  them  as  well  as  the  opportunities  for 
infection  with  fungi.  Instead,  a  tube  is  used  which  has  a  ground-glass 
cap  fitted  over  it.  This  cap  contracts  into  a  narrow  tube  plugged  with 
glass-wool;  this  plug  is  not  disturbed.  The  tube  is  cleaned,  filled,  and 
inoculated  by  removing  the  cap.  With  sufficient  opportunity  for  the 
interchange  of  air  very  little  evaporation  takes  place,  and  contamination 
of  the  culture  is  a  very  rare  occurrence.  In  inoculating  these  tubes  bits 
of  tissue  which  include  tuberculous  foci,  especially  the  most  recent,  are 
torn  from  the  organs  and  transferred  to  the  serum.  Very  little  crushing, 
if  any,  is  desirable  or  necessary.  I  think  many  failures  are  due  to  the 
often  futile  attempts  to  break  up  firm  tubercles.  Nor  should  the  bits 
of  tissue  be  rubbed  into  the  surface,  as  is  sometimes  recommended. 
After  a  stay  of  several  weeks  in  the  thermostat  I  usually  remove  the  tubes 
and  stir  about  the  bits  of  tissue.  This  frequently  is  the  occasion  for  a 
prompt  appearance  of  growth  of  tiny,  dull-grayish  colonies  within  a 
week,  as  it  seems  to  put  certain  still  microscopic  colonies  in  or  around 
the  tissues  into  better  condition  for  further  development.  From  this 
first  growth  of  tubercle  bacilli  fresh  serum  tubes  are  inoculated.  From 
these  either  serum  or  glycerin-agar  tubes  are  inoculated.  The  thermostat 
should  be  fairly  constant,  as  urged  by  Koch  in  his  classic  monograph; 
but  I  look  upon  moisture  as  of  more  importance.  If  possible  a  thermo- 
stat should  be  used  which  is  opened  only  occasionally.  Into  this  a 
larrje  dish  of  water  is  placed,  which  keeps  the  space  saturated.  Venti- 
lation should  be  restricted  to  a  minimum.  As  a  consequence,  moulds 
grow  luxuriantly,  and  even  the  gummed  labels  must  be  replaced  by 
pieces  of  stiff  manila  paper  fastened  to  the  tube  with  a  rubber  band . 


294  BACTERIA  PATHOGENIC  TO  MAN 

By  keeping  the  tubes  inclined  no  undue  amount  of  condensation  of 
water  can  collect  in  the  bottom,  and  the  upper  portion  of  the  serum 
remains  moist.  The  only  precaution  to  be  applied  to  prevent  infection 
with  moulds  is  to  thoroughly  flame  the  joint  between  the  tube  and  cap, 
as  well  as  the  plugged  end,  before  opening  the  tube/' 

In  our  experience,  when  cultures  are  made  exactly  according  to  the 
above  directions,  a  growth  is  usually  obtained,  but  Dorset  advises  the 
use  of  an  egg  medium.  Many,  including  ourselves,  have  had  good 
results  with  it.  It  is  more  difficult  to  get  a  growth  of  bovine  than  of 
the  human  type  of  bacilli.  All  methods  frequently  fail  when  the 
tuberculous  tissue  used  contains  very  few  bacilli. 

Pathogenesis. — The  tubercle  bacillus  is  pathogenic  not  only  to  man, 
but  to  a  large  number  of  animals,  such  as  the  monkey,  pig,  cow,  cat, 
etc.  Guinea-pigs  are  extremely  susceptible,  and  are  much  used  for 
the  detection  of  tubercle  bacilli  in  suspected  material.  When  inoculated 
with  the  minutest  doses  of  the  living  bacilli  they  usually  succumb  to 
the  disease.  Infection  is  most  rapidly  produced  by  intraperitoneal 
injection.  If  a  large  dose  is  given  death  follows  in  from  ten  to  twenty 
days.  The  omentum  is  found  to  be  clumped  together  in  sausage-like 
masses  and  converted  into  hard  knots,  which  contain  many  bacilli. 
There  is  no  serous  fluid  in  the  peritoneal  cavity,  but  generally  in  both 
pleural  sacs.  The  spleen  is  enlarged,  and  it,  as  well  as  the  liver  and 
peritoneum,  contains  large  numbers  of  tubercle  bacilli.  If  smaller 
doses  are  given  the  disease  is  prolonged.  The  peritoneum  and  interior 
organs — spleen,  liver,  etc. — are  then  filled  with  tubercles.  On  sub- 
cutaneous injection,  for  instance,  into  the  abdominal  wall,  there  is  a 
thickening  of  the  tissues  about  the  point  of  inoculation,  which  breaks 
down  in  one  to  three  weeks  and  leaves  a  sluggish  ulcer  covered  with 
cheesy  material.  The  neighboring  lymph  glands  are  swollen,  and  at 
the  end  of  two  or  three  weeks  may  attain  the  size  of  hazel-nuts.  Soon 
an  irregular  fever  is  set  up,  and  the  animal  becomes  emaciated,  usually 
dying  within  four  to  eight  weeks.  If  the  injected  material  contained 
only  a  small  number  of  bacilli  the  wound  at  the  point  of  inoculation 
may  heal  up  and  death  be  postponed  for  a  long  time.  On  autopsy  the 
lymphatic  glands  are  found  to  have  undergone  cheesy  degeneration; 
the  spleen  is  very  much  enlarged,  and  throughout  its  substance,  which 
is  colored  dark  red,  are  distributed  masses  of  nodules.  The  liver  is 
also  enormously  increased  in  size,  streaked  brown  and  yellow,  and  the 
lungs  are  filled  with  grayish-white  tubercles;  but,  as  a  rule,  the  kidneys 
contain  no  nodules.  Tubercle  bacilli  are  always  found  in  the  affected 
tissues,  but  the  more  chronic  the  process  the  fewer  the  bacilli  that  are 
apt  to  be  present. 

Rabbits  are  also  quite  susceptible  to  tuberculosis,  but  considerably  less 
so  than  guinea-pigs.  In  rabbits  death  almost  invariably  follows  inocu- 
lations of  tuberculous  material  into  the  anterior  chamber  of  the  eye. 
The  local  effects  are  iris  tuberculosis  and  cheesy  degeneration  of  the 
pupil.  The  bacilli  then  penetrate  to  the  neighboring  lymph  glands, 
producing  softening  of  these,  then  pulmonary  tuberculosis,  general 


THE  BACILLUS  OF  TUBERCULOSIS  295 

tuberculosis,  and  finally  death  at  the  end  of  several  weeks  or  months. 
Subcutaneous  inoculations  are  less  effective,  and  in  small  doses 
frequently  do  not  kill.  Intravenous  and  intraperitoneal  injections 
usually  produce  general  tuberculosis  and  death  at  the  end  of  a  few 
weeks. 

Of  other  susceptible  animals,  field-mice  and  cats  are  readily  infected 
by  artificial  inoculations  of  tuberculous  material;  rats,  white  mice,  and 
dogs  only  when  very  large  doses  are  given.  All  these  animals  present 
the  anatomical  lesions  of  miliary  tuberculosis.  Canaries  are  also 
susceptible  to  inoculations  of  the  tubercle  bacillus,  but  not  sparrows. 
Fowls  and  pigeons  are  only  moderately  susceptible  to  the  bacillus 
derived  from  man.  Among  the  larger  birds,  parrots  alone  would  seem 
to  be  clearly  susceptible. 

ANIMAL  INFECTION  BY  NATURAL  METHODS. — Besides  the  artificial 
modes  of  infection  referred  to,  tuberculosis  may  be  caused  in  animals 
by  feeding  them  with  tuberculous  material.  In  this  case  evidence  of 
infection  is  usually  shown  in  the  mesenteric  glands,  while  the  intestinal 
walls  are  frequently  not  affected.  Bacilli  accompanied  by  fat  much 
more  readily  pass  through  the  intestinal  mucous  membrane  or  that 
of  the  tonsils  and  pharynx.  It  is  certain  that  tuberculous  infection  may 
be  caused,  under  certain  conditions,  by  absorption  through  serous  or 
mucous  membranes  without  the  evidence  of  any  local  lesion. 

The  experimental  production  of  tuberculosis  by  inhalation  of  bacilli 
has  been  demonstrated  by  Koch  in  guinea-pigs,  rabbits,  rats,  and  mice, 
and  his  results  have  since  been  confirmed  by  many  others;  but  in  these 
experiments  the  bacilli  were  usually  inhaled  in  the  form  of  a  very  thin 
spray  in  which  they  were  suspended.  The  experimental  inhalation  of 
dry  tuberculous  dust  has  less  often  proved  successful. 

Various  other  tuberculous  affections  which  are  natural  in  man  have 
been  produced  experimentally  in  animals,  as,  for  instance,  tuberculosis 
of  the  joints,  tuberculous  abscess,  etc. 

ACTION  UPON  THE  TISSUES  OF  THE  POISONS  PRODUCED  BY  THE 
TUBERCLE  BACILLUS. — Soon  after  the  introduction  into  the  tissues  of 
tubercle  bacilli,  either  living  or  dead,  the  cells  surrounding  them  begin 
to  show  that  some  irritant  is  acting  upon  them.  The  connective-tissue 
cells  become  swollen  and  undergo  mitotic  division,  the  resultant  cells 
being  distinguished  by  their  large  size  and  pale  nuclei.  A  small  focus 
of  proliferated  epithelioid  cells  is  thus  formed  about  the  bacilli,  and 
according  to  the  intensity  of  the  inflammation  these  cells  are  surrounded 
by  a  larger  or  smaller  number  of  the  lymphoid  cells.  When  living 
bacilli  are  present  and  multiplying  the  lesions  progress,  the  central 
cells  degenerate  and  die,  and  a  cheesy  mass  results,  which  later  may 
lead  to  the  formation  of  cavities.  Dead  bacilli,  on  the  other  hand,  give 
off  sufficient  poison  to  cause  the  less  marked  changes  only,  and  never 
produce  cavities  (Prudden  and  Hodenpyl).  Of  the  gross  pathological 
lesions  produced  in  man  by  the  tubercle  bacilli  the  most  characteristic 
are  small  nodules,  called  miliary  tubercles.  When  young,  and  before 
they  have  undergone  degeneration,  these  tubercles  are  gray  and  trans- 


296  BACTERIA  PATHOGENIC  TO  MAN 

lucent  in  color,  somewhat  smaller  than  a  millet  seed  in  size,  and  hard! 
in  consistence. 

But  miliary  tubercles  are  not  the  sole  tuberculous  products.  The 
tubercle  bacilli  may  cause  the  diffuse  growth  of  a  tissue  identical  in 
structure  with  that  of  miliary  tubercles — that  is,  composed  of  a  base- 
ment substance,  containing  epithelioid,  giant,  and  lymphoid  cells. 
This  diffuse  tubercle  tissue  also  tends  to  undergo  cheesy  degeneration. 

DISTRIBUTION  OF  TUBERCLE  BACILLI  IN  THE  TISSUES. — In  acute 
tuberculosis,  especially  when  caseation  is  rapidly  spreading,  the  bacilli 
are  usually  abundant.  They  are  generally  scattered  irregularly  through 
the  tissues  or  in  small  groups.  They  are  occasionally  found  in  the 
leukocytes  and  in  the  giant  and  epithelioid  cells.  In  subacute  and 
chronic  lesions  they  are  usually  few  in  number.  Sometimes  in  old 
caseous  materials  numerous  stained  granular  points  are  seen;  these 
are  supposed  by  some  to  be  a  resting  stage  similar  to  spores,  but  not 
resistant  to  heat. 

USUAL  POINT  OF  ENTRANCE  OF  INFECTION. — Infection  by  the  tubercle 
bacillus  takes  place  usually  through  the  respiratory  tract  or  the  digestive 
tract  including  the  pharynx  and  tonsils,  more  rarely  through  wounds 
of  the  skin. 

Tuberculosis  may  be  considered  to  be  caused  chiefly  by  the  direct 
transmission  of  tubercle  bacilli  to  the  mouth  through  soiled  hands, 
lips,  handkerchiefs,  food,  etc.,  or  by  the  inhalation  of  fine  particles  of 
mucus  thrown  off  by  coughing  or  loud  speaking,  or  of  tuberculous  dust. 

TUBERCULOSIS  OF  SKIN  AND  Mucous  MEMBRANES. — When  the  skin 
or  mucous  membranes  are  superficially  infected  through  wounds  there 
may  develop  lupus,  ulceration,  or  a  nodular  growth.  The  latter  two 
forms  of  infection  are  apt  after  an  interval  to  cause  the  involvement  of 
the  nearest  lymphatic  glands,  and  then  finally  the  deeper  structures. 

TUBERCULOSIS  OF  DIGESTIVE  TRACT. — Tuberculosis  of  the  gums,, 
cheeks,  and  tongue  are  rare,  and  usually  occur  through  the  germs  enter- 
ing lacerations  caused  by  sharp,  ragged  teeth.  The  tonsils  and  pharynx 
are  somewhat  more  often  involved.  The  stomach  protected  through 
its  acid  gastric  juice,  and  oesophagus  by  its  epithelium,  are  almost  never 
attacked.  The  small  intestines,  rich  in  lymph  glands,  are  rather  fre- 
quently the  seat  of  infection  from  bacilli  swallowed  with  the  food.  In 
a  striking  case  four  previously  healthy  children  died  within  a  short 
period  of  one  another.  Their  nurse  was  found  to  have  tuberculosis 
of  the  antrum  of  Highmore,  with  a  fistulotis  opening  into  the  mouth. 
She  had  the  habit  of  putting  the  spoon  with  which  she  fed  the  children 
into  her  mouth  so  as  to  taste  the  food  before  it  was  given  to  them.  As 
already  noted,  the  bacilli  frequently  pass  through  the  mucous  mem- 
brane to  the  lymph  glands  without  leaving  any  lesions. 

Intestinal  and  mesenteric  tuberculosis,  which  is  rather  common  witb 
children,  is  due  not  only  to  swallowing  the  bacilli  received  in  the  above- 
ways,  from  human  sources,  but  also  to  the  ingestion  of  tuberculous  milk. 

TUBERCULOSIS  OF  RESPIRATORY  TRACT.— The  lungs  are  the  most 
frequent  location  of  tuberculous  inflammation,  in  spite  of  the  fact  that 


TUJ-:  BACILLUS  OF  TUBERCULOSIS  297 

on  account  of  their  location  they  are  greatly  protected.  Most  of  the  bacilli 
are  caught  upon  the  nasal  or  pharyngeal  mucous  membranes.  Only 
a  small  percentage  can  find  their  way  to  the  larynx  and  trachea,  and 
still  less  to  the  smaller  bronchioles.  From  the  examination  of  the  lungs 
of  miners  as  well  as  from  experimental  tests  there  is  no  doubt  but  that 
some  of  the  bacilli  find  their  way  into  the  deeper  bronchi.  The  deeper 
the  bacilli  penetrate  the  more  unlikely  that  they  can  be  cast  out.  The 
healthy  lung  tissue  usually  destroys  these  bacilli.  The  nasal  cavities 
are  rarely  affected  with  tuberculosis,  but  more  often  the  retropharyn- 
geal  tissue.  Tuberculosis  of  this  tissue  as  well  as  that  of  the  tonsils  is 
apt  to  give  rise  to  infection  of  the  lymphatic  glands  of  the  neck.  It 
is  believed  that  just  as  bacilli  may  pass  through  the  intestinal  walls  to 
infect  the  mesenteric  glands  so  bacilli  may,  without  leaving  any  trace, 
pass  through  the  tonsils  to  the  glands  of  the  neck.  It  is  now  well 
established  that  infection  taking  place  through  various  channels  may 
find  its  way  to  the  lungs  and  excite  there  the  most  extensive  lesions. 

TUBERCULOSIS  OF  LARYNX. — Primary  infection  of  the  larynx  is  rare. 
Secondary  infection  is  fairly  common.  The  region  of  the  vocal  cords 
and  the  interarytenoid  space  are  the  special  sites  attacked. 

INFECTION  BY  INHALATION  OF  DRIED  AND  MOIST  BACILLI. — The 
most  common  mode  of  infection  is  by  means  of  tuberculous  sputum, 
which,  being  coughed  up  by  consumptives,  is  either  disseminated  as  a 
fine  spray  and  so  inhaled,  or,  carelessly  expectorated,  dries  and  distributes 
numerous  virulent  bacilli  in  the  dust.  As  long  as  the  sputum  remains 
moist  there  is  no  danger  of  dust  infection,  but  only  of  direct  contact; 
it  is  only  when  it  becomes  dry,  as  on  handkerchiefs,  bedclothes,  and  the 
floor,  etc.,  that  the  dust  is  a  source  of  danger. 

A  great  number  of  the  expectorated  and  dried  tubercle  bacilli 
undoubtedly  die,  especially  when  exposed  to  the  action  of  direct 
sunlight;  but  when  it  is  considered  that  as  many  as  five  billion 
virulent  tubercle  bacilli  may  be  expectorated  by  a  single  tuberculous 
individual  in  twenty-four  hours,  it  is  evident  that  even  a  much  smaller 
proportion  than  are  known  to  stay  alive  will  suffice  in  the  immediate 
vicinity  of  consumptives  to  produce  infection  unless  precautions  are 
taken  to  prevent  it.  The  danger  of  infection  is  greatest,  of  course,  in 
the  close  neighborhood  of  tuberculous  patients  who  expectorate  pro- 
fusely and  indiscriminately — that  is,  without  taking  the  necessary 
means  for  preventing  infection.  There  is  much  less  danger  of  infection 
at  a  distance,  as  in  the  streets,  for  instance,  where  the  tubercle  bacilli 
have  become  so  diluted  that  they  are  less  to  be  feared.  In  rooms  the 
sputum  is  not  only  protected  from  the  direct  sunlight,  but  it  is  con- 
stantly broken  up  and  blown  about  by  the  walking,  closing  of  doors, 
etc.  In  crowded  streets  on  windy  days  infected  dust  must  sometimes 
be  in  the  air  unless  the  expectoration  of  consumptives  is  controlled. 

Exhaustive  experiments  made  by  many  observers  have  shown  that 
particles  of  dust  collected  from  the  immediate  neighborhood  of  con- 
sumptives, when  inoculated  into  guinea-pigs,  produce  tuberculosis 
in  a  considerable  percentage  of  them;  whereas,  the  dust  from  rooms 


298  BACTERIA  PATHOGENIC  TO  MAN 

inhabited  by  healthy  persons  or  the  dusts  of  the  streets  does  so  only  in 
an  extremely  small  percentage.  Fliigge  is  probably  right  in  thinking  that 
the  dust  which  is  fine  enough  to  remain  for  a  long  time  in  suspension 
in  the  air  is  usually  free  from  living  bacilli.  It  is  in  the  coarser  though  still 
minute  particles,  those  in  which  the  bacilli  are  protected  by  an  envelope 
of  mucus, that  the  germs  resist  drying  for  considerable  periods.  These  are 
carried  only  short  distances  by  air  currents.  Such  results  as  those  ob- 
tained by  Straus,  who  on  examining  the  nasal  secretions  of  twenty-nine 
healthy  persons  living  in  a  hospital  with  consumptive  patients,  found 
tubercle  bacilli  in  nine  of  them,  must  be  accepted  with  some  reserve, 
since  we  know  that  in  the  air  there  are  bacilli  which  look  and  stain  like 
tubercle  bacilli  and  yet  are  totally  different.  It  may  be  said  that  the 
danger  of  infection  from  slight  contact  with  tuberculosis  is  not  so  great 
as  it  is  considered  by  many,  but  that  on  this  account  it  is  all  the  more  to 
be  feared  and  guarded  against  in  the  immediate  neighborhood  of  con- 
sumptives. Those  who  are  most  liable  to  infection  from  this  source 
are  the  families,  the  nurses,  the  fellow-workmen,  and  fellow-prisoners 
of  persons  suffering  from  the  disease.  In  this  connection,  also,  atten- 
tion may  be  drawn  to  the  fact  that  rooms  which  have  been  recently 
occupied  by  consumptives  are  not  infrequently  the  means  of  producing 
infection  (as  has  been  clinically  and  experimentally  demonstrated) 
from  the  deposition  of  tuberculous  dust  on  furniture,  walls,  floors,  etc. 
Flugge  has  recently  drawn  attention  to  the  fact  that  in  coughing,  sneez- 
ing, etc.,  very  fine  particles  of  throat  secretion  are  thrown  out  and 
carried  by  air  currents  many  feet  from  the  patient  and  remain  sus- 
pended in  the  air  for  a  considerable  time.  This  is  another  means  of 
infection,  but  probably  a  less  frequent  one.  To  encourage  us  we  now 
have  a  mass  of  facts  which  go  to  show  that  when  the  sputum  is  care- 
fully looked  after  there  is  very  little  danger  of  infecting  others  except 
by  close  personal  contact. 

INDIVIDUAL  SUSCEPTIBILITY. — It  is  believed  by  many  that  in  demon- 
strating the  possibility  of  infection  in  pulmonary  tuberculosis  its  occur- 
rence is  sufficiently  explained;  but  they  leave  out  another  and  most 
important  factor  in  the  production  of  an  infectious  disease — individual 
susceptibility.  That  this  susceptibility,  or  "predisposition/'  as  it  is 
improperly  called,  may  be  either  inherited  or  acquired  is  now  an  accepted 
fact  in  medicine.  It  is  even  thought  that  the  physical  signs  and  char- 
acters— the  phthisical  habit — which  indicate  this  susceptibility  can  be 
externally  recognized.  At  first  the  inherited  susceptibility  was  con- 
sidered more  important  than  the  acquired,  but  now  much  that  was 
attributed  to  the  former  is  known  to  be  explained  by  the  fact  of  living 
in  an  infected  area.  The  acquired  susceptibility  may  arise  from 
faulty  ^physical  development  or  from  depression,  sickness,  overwork, 
excessive  use  of  alcohol,  etc.  Unquestionably,  vast  differences  exist 
in  different  individuals  in  the  intensity  of  the  tuberculous  process  in 
the  lung.  That  this  does  not  depend  chiefly  upon  a  difference  in  viru- 
lence of  the  infection  is  evident,  from  the  fact  that  individuals  con- 
tracting tuberculosis  from  the  same  source  are  attacked  with  different 


Till-:   BACILLUS  OF  TUBERCULOSIS  299 

severity,  and  that  there  is,  as  a  rule,  no  great  difference  in  degrees  of 
virulence  for  animals  in  the  tubercle  bacilli  obtained  from  different 
sources.  As  is  seen  from  the  results  of  post-mortem  examinations  in 
which,  according  to  the  completeness  of  the  examinations,  the  remains 
of  old  tuberculous  processes  have  been  found  in  the  lungs  of  about 
one-third  to  one-half  of  all  the  bodies  examined,  many  cases  of  pul- 
monary infection  must  occur  without  showing  any  visible  evidence  of 
disease,  and  heal  of  their  own  accord.  The  possibility  of  favorably 
influencing  in  many  an  existing  tuberculosis  by  treatment  also  proves 
that,  under  natural  conditions,  there  is  a  varying  susceptibility  to  the  dis- 
ease. Clinical  experience  teaches,  likewise,  that  good  hygienic  con- 
ditions, pure  air,  good  food,  freedom  from  care,  etc.,  increase  immunity 
to  phthisis.  Animal  experiments  have  shown  that  not  only  are  there 
differences  of  susceptibility  in  various  animal  species,  but  also  an 
individual  susceptibility  in  the  same  species.  The  doctrine  of  indi- 
vidual susceptibility,  therefore,  is  seen  to  be  founded  on  fact,  although 
the  reasons  for  it  are  only  partially  understood. 

INFECTION  BY  INGESTION  OF  MILK  AND  MEAT. — Phthisical  sputum, 
however,  is  not  held  responsible  for  the  occurrence  of  all  human  tuber- 
culosis. Milk  also  serves  as  a  conveyer  of  infection,  whether  it  be  the 
milk  of  nursing  mothers  suffering  from  consumption  or  the  milk  of 
tuberculous  cows.  The  transmission  of  tubercle  bacilli  in  the  milk  of 
tuberculous  cows  has  been  abundantly  proved.  Formerly  it  was  thought 
that  in  order  to  produce  infection  by  milk  there  must  be  a  local  tuber- 
culous affection  of  the  udder;  but  it  is  now  known  that  tubercle  bacilli 
may  be  found  in  the  milk  when  an  internal  organ  is  infected,  and  when 
careful  search  fails  to  detect  any  udder  disease.  The  milk  of  every  cow 
which  has  any  well-developed,  internal,  tuberculous  infection  must  there- 
fore be  considered  as  possibly  containing  tubercle  bacilli.  Rabino- 
witsch  and  Kempner  proved  beyond  all  question  that  not  only  the  milk 
of  tuberculous  cattle,  which  showed  no  appreciable  udder  disease,  but 
also  those  in  which  tuberculosis  wras  only  detected  through  tuberculin, 
frequently  contained  tubercle  bacilli.  Different  observers  have  found 
tubercle  bacilli  in  the  milk  of  from  20  to  60  per  cent,  of  tuberculous 
cows.  When  we  consider  the  prevalence  of  tuberculosis  among  cattle 
we  can  readily  realize,  even  if  the  bovine  bacillus  with  difficulty  infects 
human  beings,  the  danger  to  which  children  are  exposed  from  this 
source  of  infection.  Thus,  taking  the  abattoir  statistics  of  various 
countries,  we  find  that  about  10  per  cent,  of  the  cattle  slaughtered 
were  tuberculous.  A  less  probable  source  of  infection  by  way  of 
the  intestines  is  the  flesh  of  tuberculous  cattle.  Here  the  danger  is 
considerably  less,  from  the  fact  that  meat  is  usually  cooked,  and  also 
because  the  muscular  tissues  are  seldom  attacked.  In  view  of  the 
finding  of  the  bovine  type  of  bacilli  in  a  considerable  percentage  of 
the  few  cases  tested  of  tuberculous  children,  the  legislative  control 
and  inspection  of  cattle  and  milk  would  seem  to  be  an  absolute  neces- 
sity. As  a  practical  and  simple  method  of  preventing  infection  from 
suspected  milk  the  sterilization  (by  heat)  of  the  milk  used  as  food  must 


300  BACTERIA  PATHOGENIC  TO  MAN 

commend  itself  to  all.  It  is  only  right  to  state,  however,  that  up  to 
the  present  time  the  actual  proof  that  human  tuberculosis  has  fre- 
quently come  from  milk  or  food  infected  with  bovine  tuberculosis  is 
very  small,  and  that  it  is  probable  that  the  bovine  bacilli  are  not  as 
virulent  for  man  as  for  animals.  The  relation  of  bovine  to  human 
tubercle  bacilli  will  be  discussed  more  fully  later. 

AUTOINFECTION  BY  SWALLOWING  SPUTUM. — The  secondary  forms 
of  tuberculosis  which  often  succeed  a  primary  infection  of  the  lungs 
may  be  explained  as  autoinfections,  from  the  coughing  up  and  swal- 
lowing of  sputum  containing  bacilli.  It  is  a  wonder,  indeed,  that  intes- 
tinal tuberculosis  is  not  more  common  than  it  is  in  consumption;  but 
this  is  probably  due  to  the  action  of  the  gastric  juice  and  to  the  fact 
that  in  adults  the  intestines  are  comparatively  insusceptible. 

Hypothesis  of  Transmissibility  of  Tubercle  Bacilli  to  the  Foetus. — There 
is  some  evidence  of  the  transmission  of  tubercle  bacilli  from  the  mother 
to  the  foetus  in  animals.  The  first  authentic  case  recorded  is  that 
reported  by  Johne  of  an  eight-months-old  calf  foetus;  other  cases  have 
since  been  reported.  With  regard  to  tuberculosis  in  the  human  foetus 
the  evidence  is  not  so  clear,  though  some  twenty  cases  have  been  reported 
of  tuberculosis  in  newly  born  infants,  and  about  a  dozen  cases  are 
recorded  of  placental  tuberculosis.  The  fact  that  statistics  show  a 
greater  frequency  of  tuberculous  diseases  in  children  during  the  first 
than  in  the  following  years  of  life,  does  not  strengthen  the  hypothesis 
of  frequent  infection  in  utero;  for  nursing  infants  would  naturally  be 
more  exposed  to  infection  through  the  mother's  milk  and  through 
personal  contact  than  others.  According  to  experiments  upon  laboratory 
animals  one  would  expect  to  find  in  man  fetal  or  placental  tuberculous 
infection  more  common  than  it  is,  whereas  it  is  extremely  rare,  even 
if  the  few  cases  reported  be  accepted  as  proven.  Possibly  the  few 
bacilli  which  may  be  transmitted  to  the  foetus  do  not  find  conditions 
favorable  for  their  development,  and,  being  so  few  in  number,  die;  or 
they  may  remain  latent,  as  has  been  suggested  by  Behring  and  others, 
for  certain  lengths  of  time  without  producing  visible  effects,  and  only 
show  symptoms  of  infection  later;  but  we  have  no  experimental  con- 
firmation of  any  such  latency  existing  with  regard  to  the  tubercle  bacillus, 
and  it  is  not  to  be  assumed  that  it  does  exist.  As  to  the  infection  of  the 
foetus  from  the  paternal  side,  where  the  father  has  tuberculosis  of  the 
scrotum  or  seminal  vessels  (which  have  been  found  to  be  tuberculous 
in  exceptional  cases),  we  have  no  reason  to  suppose  that  such  can  occur. 
There  are,  however,  grounds  for  belief  that  infection  in  this  way  may 
take  place  from  husband  to  wife.  Thus,  Gartner  found,  as  a  result  of 
his  experiments  in  animals,  that  a  large  majority  of  the  guinea-pigs 
and  rabbits  which  were  brought  together  with  males  whose  semen 
contained  tubercle  bacilli  died  of  primary  genital  tuberculosis;  but 
from  the  rarity  of  this  affection  in  women  and  cows  it  may  be  assumed 
that  tubercle  bacilli  occur  very  much  less  frequently  in  semen  of  men 
and  cattle  than  in  that  of  the  smaller  animals.  It  is  believed  that  the 
semen  of  those  suffering  from  advanced  or  local  genital  tuberculosis 


^rcr-yVJ^X 
THE  BACILLUS  OF~  TUBERCULOSIS  301 

contain  tuberculous  toxins  which  cripple  the  activity  of  the  sperma- 
tozoa. 

Length  of  Time  Tubercle  Bacilli  Remain  Virulent  in  Sputum. — According 
to  experimental  investigations,  the  virulence  of  dried  tuberculous  sputum 
is  not  suddenly  but  gradually  lost,  a  certain  proportion  of  it  retaining 
its  specific  infective  power  under  ordinary  conditions,  as  in  a  dwelling- 
room,  for  at  least  two  or  three  months,  and  occasionally  for  a  year  or 
more. 

Attenuation. — Tul>ercle  bacilli  when  subjected  to  deleterious  influences 
or  to  growth  on  culture  media  gradually  lose  their  virulence.  A  culture 
which  Trudeau  has  grown  on  suitable  media  for  ten  years  is  no  longer 
capable  of  causing  tuberculosis  in  healthy  guinea-pigs,  although  origi- 
nally virulent.  Cultures  grown  at  temperatures  of  42°  C.  become  atten- 
uated more  quickly. 

Mixed  Infection. — In  regions  where  tuberculous  processes  are  on  the 
surface,  such  as  lung  and  skin  infections,  and  also  when  the  infection 
itself  is  multiple,  as  in  disease  of  the  glands  of  the  neck  from  tonsillar 
absorption,  the  tubercle  bacilli  are  usually  associated  with  one  or  more 
other  varieties  of  organisms.  Those  of  most  importance  are  th'e  strepto- 
coccus, pneumococcus,  and  influenza  bacillus.  Besides  these  many 
other  varieties  are  met  with  occasionally  in  individual  cases.  What  the 
influence  of  this  secondary  or  mixed  infection  is,  under  all  circumstances, 
is  not  exactly  known;  but  generally  the  effect  is  an  unfavorable  one, 
and  not  infrequently  after  a  time  the  disease  takes  oh  a  septicaemic 
character.  For  the  technique  employed  in  examining  sputa  for  mixed 
infection  see  page  312. 

Immunization. — As  in  other  infectious  diseases,  many  attempts  have 
been  made  to  produce  an  artificial  immunity  against  tuberculosis,  but 
so  far  the  results  have  been  only  fairly  satisfactory.  The  great  majority 
of  mankind  has  in  a  varying  degree  a  natural  immunity  against  tuber- 
culosis. In  many  individuals  this  immunity  is  only  relative,  and  is 
maintained  only  so  long  as  the  health  is  kept  at  a  high  standard  or 
the  exposure  to  infection  not  too  intense  or  prolonged.  An  unfavorable 
environment,  the  occurrence  of  some  other  infectious  disease,  overwork, 
dissipation,  or,  in  fact,  anything  which  tends  to  depreciate  the  nutrition 
of  the  body,  is  apt  to  render  the  individual  previously  immune  susceptible 
to  the  tubercle  bacillus. 

Acquired  immunity  against  many  bacterial  diseases  is  acquired 
within  a  few  days  or  weeks  after  the  development  of  infection.  This 
immunity  may  be  complete  or  slight  and  vary  greatly  in  its  dura- 
tion. There  is  little  in  the  clinical  history  of  tuberculosis  which 
shows  that  acquired  immunity  occurs  in  this  disease,  for  relapse  is  the 
rule,  and  one  attack  does  not  seem  to  afford  any  protection  against  a 
later  one.  For  this  reason  the  production  of  an  artificial  immunity 
against  tuberculosis  has  always  been  looked  upon  as  a  result  possibly 
never  to  be  achieved.  The  careful  study  of  tuberculosis  seems,  how- 
ever, to  indicate  an  attempt  on  the  part  of  nature  at  the  production  of 
acquired  immunity  in  this  disease.  It  is  thought  that  from  30  to  60 


302  BACTERIA  PATHOGENIC  TO  MAN 

per  cent,  of  cadavers  show  the  healed  lesions  of  tuberculosis.  The 
small  proportion  of  these  which  progressed  to  serious  lesions  or  became 
reinfected  indicate  a  degree  of  acquired  immunity.  Artificial  im- 
munity is  an  attempt  to  imitate  nature's  methods,  and  is  obtained 
by  the  inoculation  of  a  modified  living  culture  or  of  toxins  and  dead 
bacteria.  The  injection  of  toxins,  as  in  Koch's  tuberculin  treatment, 
produces  in  animals  a  certain  degree  of  acquired  resistance  to  larger 
doses  of  toxins,  but  does  not  protect  to  any  appreciable  degree  from 
subsequent  living  tubercle  bacilli,  or  produce  in  animals  an  antitoxic 
serum.  In  1892  Trudeau  succeeded  in  producing  in  rabbits  an  appre- 
ciable immunity  by  inoculations  of  living  avian  cultures.  The  rabbits 
so  treated  supported,  as  a  rule,  inoculation  of  virulent  tubercle  bacilli 
in  the  anterior  chamber  of  the  eye,  while  in  controls  the  eyes  were 
invariably  lost.  Later,  attenuated  human  cultures  were  used  with  the 
same  results.  De  Schweinitz,  McFadyan,  Behring,  and  Pearson  Gilli- 
land  have  since  reported  successful  results.  The  latter  two  treated  a 
number  of  cows  by  giving  each  of  them  seven  intravenous  injections  of 
1  to  6  c.c.  of  an  emulsion  of  tubercle  bacilli.  This  was  of  an  opacity 
equal  to  a  twenty-four-hour  broth  culture  of  typhoid  bacilli.  They 
report  from  their  investigations1  that  the  treatment  had  the  effect  not 
only  in  keeping  in  check  the  progress  of  the  tuberculous  process,  but 
of  causing  in  some  cases  a  distinct  retrogression.  The  bacilli  remained 
alive  in  the  encapsulated  lesions. 

The  work  already  done  is  believed  by  Trudeau  to  establish  the 
principle  that  in  order  to  be  successful  the  protective  inoculation  must 
be  made  with  living  germs  of  such  diminished  virulence  for  the  animal 
experimented  upon  as  to  produce  a  reaction  ending  in  healing  of  the 
process  at  first  set  up  by  them.  This  is  termed  by  Behring  isopathic 
immunity. 

The  avian  and  bovine  bacilli  immunize  against  infection  from  human 
bacilli  equally  as  well  as  the  attenuated  human  variety.  This  is  strong 
evidence  in  favor  of  the  genetic  unity  of  all  tubercle  bacilli.  Up  to 
the  present  time  the  results  in  animals  hardly  permit  the  inoculation 
of  man  with  living  bacilli  for  purposes  of  producing  immunity.  The 
practical  difficulties  which  confront  one  make  it  at  present  probably 
unadvisable  to  use  such  methods  in  cows  except  in  an  experimental 
way. 

The  serum  of  animals  treated  with  bacilli  and  their  products  has 
not  given  curative  results. 

Among  the  numerous  medicinal  agents  that  have  been  tried  without 
avail  to  protect  animals  against  the  action  of  the  tubercle  bacillus 
may  be  mentioned  tannin,  menthol,  sulphuretted  hydrogen,  mercuric 
chloride,  creosote,  creolin,  phenol,  arsenic,  eucalyptol,  etc. 

Agglutination. — The  results  obtained  by  various  observers  has  been 
very  conflicting.  Two  methods  are  employed  in  making  the  test.  In 
one  a  vigorous  growth  of  bacilli  is  dried,  ground  up  and  an  emulsion 

i  University  of  Pennsylvania  Medical  Bulletin,  April,  1905. 


THE  BACILLUS  OF  TUBERCULOSIS  303 

made.  In  the  other  Arloing  and  Courmont  grow  the  culture  for  a 
time  on  potato  and  then  in  bouillon.  In  this  way  a  homogeneous 
culture  of  separate  bacilli  is  obtained  which  can  be  used  for  agglutina- 
tion. The  examination  is  usually  made  macroscopically,  and  requires 
twelve  to  twenty-four  hours.  At  present  the  test  cannot  be  advised  as 
useful  in  diagnosis  as  the  sera  of  cases  suffering  from  tuberculosis  fre- 
quently fail  to  give  a  reaction,  while  the  sera  from  those  having  no 
detectable  tuberculosis  frequently  cause  a  good  reaction.  A  positive 
agglutination  test  in  tuberculosis  .^eems  to  be  a  favorable  sign  as  indi- 
cating resistance  to  infection  by  the  body. 

Chemical  Constituents  of  Tubercle  Bacilli. — The  bacilli  contain  on  an 
average  86  per  cent,  water.  The  dry  substance  consists  of  material 
soluble  in  alcohol  and  ether,  of  proteid  substance  extracted  by  warm 
alkaline  solutions,  and  of  carbohydrates  and  ash.  The  alcohol-ether 
extract  equals  about  one-quarter  of  the  dry  substance  and  consists  of 
15  per  cent,  of  a  fatty  acid,  which  is  mostly  combined  with  an  alcohol 
to  make  a  wax.  No  glycerin  is  present  and,  therefore,  no  true  fat.  It 
is  on  the  presence  of  this  wax  that  the  staining  characteristics  depend. 
Other  substances  produce  abscess,  necrosis,  and  cheesy  degeneration. 
Lecithin  and  a  convulsive  poison  are  also  present  in  the  extract. 

The  substances  left  after  the  ether-alcohol  extraction  are  mostly 
proteid  substances.  A  nucleic  acid  which  contains  phosphorus  is  present 
which,  according  to  Behring,  is  the  specific  principle  of  tuberculin. 

Tuberculin  (Koch's). — Tuberculin  contains  not  only  the  products  of 
the  growth  of  the  tubercle  bacilli  in  the  nutrient  bouillon  which  with- 
stand heat  as  well  as  substances  extracted  from  the  bodies  of  the  bacilli 
themselves,  but  also  the  materials  originally  contained  in  the  bouillon, 
which  have  remained  unaffected  by  the  activities  of  the  bacilli.  There 
are  two  preparations  known  respectively  as  the  old  and  the  new 
tuberculin. 

Old  tuberculin  is  prepared  as  follows:  The  tubercle  bacillus  is  culti- 
vated in  an  infusion  of  calf's  flesh  or  of  beef  flesh,  or  extract  to  which 
1  per  cent,  of  peptone  and  4  to  5  per  cent,  of  glycerin  have  been  added, 
the  culture  liquid  being  slightly  alkaline.  The  inoculation  is  made 
upon  the  surface  from  a  piece  of  very  thin  pellicle  from  a  young  bouillon 
culture,  or,  if  the  bouillon  culture  is  unobtainable,  with  small  masses 
from  a  culture  on  glycerin  agar.  These  masses,  floating  on  the  surface, 
give  rise  in  from  three  to  six  weeks,  according  to  the  rapidity  with 
which  the  culture  grows,  to  an  abundant  development  and  to  the  forma- 
tion of  a  tolerably  thick  and  dry,  white  crumpled  layer,  which  finally 
covers  the  entire  surface.  At  the'end  of  four  to  eight  weeks  development 
ceases,  and  the  layer  after  a  time  sinks  to  the  bottom.  Fully  developed 
cultures,  after  having  been  tested  for  purity  by  a  microscopic  exami- 
nation, are  passed  into  a  suitable  vessel  and  evaporated  to  one-tenth 
of  their  original  bulk  over  a  water-bath  at  a  temperature  of  70°  to 
80°  C.  The  liquid  is  then  filtered  through  chemically  pure  sterilized 
filter  paper.  The  crude  tuberculin  thus  obtained  contains  40  to  50  per 
cent,  of  glycerin,  10  per  cent,  of  albumoses,  traces  of  peptone,  extractives, 


304  BACTERIA  PATHOGENIC  TO  MAN 

and  inorganic  salts.  The  true  nature  of  the  toxic  substances  is  not  known. 
It  keeps  well,  retaining  its  activity  indefinitely. 

The  method  of  treatment  and  the  results  obtained  from  the  old 
tuberculin  have  been  described  by  Koch  briefly  as  follows:  After  each 
injection,  which  should  be  large  enough  to  cause  a  slight  but  not  a 
great  rise  of  temperature,  a  noticeable  improvement  in  the  tuberculous 
process  results.  The  amount  of  tuberculin  injected  is  continually 
increased,  so  as  to  continue  the  moderate  reactions.  After  several 
months  all  reactions  cease,  the  patients  having  become  temporarily 
immune  to  the  toxin,  but  not  to  the  growth  of  the  bacillus.  Further 
injections  are  now  useless  until  this  immunity  has  passed.  During  the 
treatment  the  bacilli  themselves  have  not  been  directly  affected,  and 
when  the  treatment  is  interrupted  the  tuberculous  process  is  apt  to 
progress.  Some  cases,  however,  of  pure  tuberculosis  of  moderate 
extent  become  cured  or  greatly  benefited  by  several  periods  of  treatment. 
When  the  seat  of  tuberculous  lesions  is  visible,  as  in  lupus,  a  moderate 
dose  of  tuberculin  causes  a  visible  inflammatory  reaction,  which  may 
result  in  necrosis  and  a  casting  off  of  the  infected  tissue.  The  bacilli 
themselves  are  not  killed. 

According  to  Koch,  the  substances  produced  in  the  body  by  the  old 
tuberculin  neutralized  the  tuberculous  toxins,  but  were  not  bactericidal. 
After  a  series  of  experiments  he  considered  the  difficulty  to  be  due  to 
the  nature  of  the  envelope  of  the  tubercle  bacillus,  which  made  it  difficult 
to  obtain  the  substance  of  the  bacilli  in  soluble  form  without  so  altering 
it  by  heat  or  chemicals  that  it  was  useless  to  produce  immunizing 
substances.  He  conceived  that  immunity  was  not  produced  in  man 
for  somewhat  similar  reasons — possibly  the  bacilli  never  giving  out 
sufficient  toxin  to  cause  curative  substances  to  be  produced.  He  there- 
fore decided  to  grind  up  the  dried  bacilli  and  soak  them  in  water,  and 
thus  obtain,  if  possible,  without  the  addition  of  heat,  a  soluble  extract 
of  the  body  substance  of  the  bacilli,  which  he  hoped  would  be  immun- 
izing. He  also  tried  to  eliminate  as  much  as  possible  of  the  toxic  prod- 
ucts which  produce  fever.  Buchner  by  a  different  method,  through 
crushing  under  a  great  pressure  tubercle  bacilli  mixed  with  sand, 
and  thus  squeezing  out  their  protoplasm,  obtained  a  very  similar 
•substance. 

The  new  tuberculin  formed  by  either  of  these  methods  is  a  watery 
extract  of  the  soluble  portions  of  the  unaltered  tubercle  bacilli.  As 
can  be  readily  seen,  in  a  preparation  thus  made,  contamination  is  difficult 
to  avoid,  freedom  from  intact  bacilli  is  uncertain,  and  the  strength  of 
the  solution  prepared  at  different  times  is  variable.  Twenty  per  cent, 
of  glycerin  is  added  to  preserve  the  tuberculin  from  contamination. 
After  three  years  of  trial  the  results  obtained  with  the  new  tuberculin 
preparations  cannot  be  considered  to  have  exerted  either  very  different 
or  very  superior  effects  to  the  older  product. 

As  to  the  results  obtained  in  general  the  reports  are  as  yet  conflicting. 
Lupus  seems  to  be  decidedly  benefited  for  a  time  both  by  the  old  and 
the  new  tuberculin.  Relapses  are,  however,  common.  On  advanced 


THE  BACILLUS  OF  TUBERCULOSIS  305 

phthisis,  laryngeal  tuberculosis,  and  other  tuberculous  processes  no 
effects  have  been  noted,  and  nearly  everyone  disapproves  of  their  use 
in  these  cases  as  well  as  in  those  where  mixed  infection  is  suspected; 
even  in  cases  of  beginning  infection,  opinions,  as  a  whole,  are  not  very 
enthusiastic.  The  new  tuberculin,  unless  prepared  with  great  care  or 
from  tubercle  bacilli,  which  are  non-virulent  for  man,  is  apt  to  be  a 
dangerous  substance.  Trudeau,  Baldwin,  and  others  found  that  with 
the  first  product  sent  out  guinea-pigs  injected  with  it  not  only  did  not 
become  immunized,  but  actually  became  infected  from  the  living  bacilli 
in  the  fluid. 

Diagnostic  Use  of  Tuberculin. — The  chief  use  to  which  tuberculin  has 
been  put  is  as  an  aid  to  the  diagnosis  of  tuberculosis  in  cattle  and  human 
beings,  and  for  this  purpose  it  has  proved  to  be  of  inestimable  value. 
Numerous  experiments  made  by  veterinary  surgeons  show  that  the 
injection  of  tuberculin  in  tuberculous  cows  in  doses  of  25  to  50 
centigrams  produces  in  at  least  95  per  cent,  a  rise  of  temperature  of 
from  1°  to  3°  C.  (2°  to  5°  F.).  The  febrile  reaction  occurs  in  from 
twelve  to  fifteen  hours  after  the  injection.  Its  intensity  and  duration 
do  not  entirely  depend  upon  the  extent  of  the  tuberculous  lesions, 
being  even  more  marked  when  these  are  slight  than  in  advanced  cases. 
In  non-tuberculous  animals  no  reaction  occurs,  or  one  much  less  than 
in  tuberculous  animals,  and  the  results  obtained  on  autopsy  justify 
the  suspicion  that  tuberculosis  exists  if  an  elevation  of  temperature  of 
a  degree  or  more  centigrade  occurs  and  remains  for  ten  hours  from 
the  subcutaneous  injection  of  the  dose  mentioned.  It  must  always  be 
remembered  that  cattle  may  have  a  rise  of  temperature  from  other 
conditions,  and  it  is  only  when  due  to  tuberculin  that  infection  is  proved. 
When  properly  carried  out  an  error  of  more  than  5  per  cent,  is  impossible. 
For  these  injections  the  original  tuberculin  is  used,  which  for  the  con- 
venience of  administration  is  diluted  with  water. 

United  States  Government  Directions  for  Inspecting  Herds  for  Tubercu- 
losis.— "Inspections  should  be  carried  on  while  the  herd  is  stabled.  If 
it  is  necessary  to  stable  animals  under  unusual  conditions  or  among 
surroundings  that  make  them  uneasy  and  excited,  the  tuberculin  test 
should  be  postponed  until  the  cattle  have  become  accustomed  to  the 
conditions  they  are  subjected  to,  and  then  begin  with  a  careful  physical 
examination  of  each  animal.  This  is  essential,  because  in  some  severe 
cases  of  tuberculosis,  on  account  of  saturation  with  toxins,  no  reaction 
follows  the  injection  of  tuberculin,  but  experience  has  shown  that  these 
cases  can  be  discovered  by  physical  examination.  This  should  include 
a  careful  examination  of  the  udder  and  of  the  superficial  lymphatic 
glands,  and  auscultation  of  the  lungs. 

"Each  animal  should  be  numbered  or  described  in  such  a  way  that 
it  can  be  recognized  without  difficulty.  It  is  well  to  number  the  stalls 
with  chalk  and  transfer  these  numbers  to  the  temperature-sheet,  so 
that  the  temperature  of  each  animal  can  be  recorded  in  its  appropriate 
place  without  danger  of  confusion.  The  following  procedure  has  been 
used  extensively  and  lias  given  excellent  results: 

20 


306  BACTERIA  PATHOGENIC  TO  MAN 

"(a)  Take  the  temperature  of  each  animal  to  be  tested  at  least 
twice,  at  intervals  of  three  hours,  before  tuberculin  is  injected. 

"(ft)  Inject  the  tuberculin  in  the  evening,  preferably  between  the 
hours  of  six  and  nine.  The  injection  should  be  made  with  a  carefully 
sterilized  hypodermic  syringe.  The  most  convenient  point  for  injec- 
tion is  back  of  the  left  scapula.  Prior  to  the  injection  the  skin  should 
be  washed  carefully  with  a  5  per  cent,  solution  of  carbolic  acid  or 
other  antiseptic. 

"  (c)  The  temperature  should  be  taken  nine  hours  after  the  injection, 
and  temperature  measurements  repeated  at  regular  intervals  of  two- 
or  three  hours  until  the  sixteenth  hour  after  the  injection. 

"  (d)  When  there  is  no  elevation  of  temperature  at  this  time  (sixteen 
hours  after  the  injection)  the  examination  may  be  discontinued;  but 
if  the  temperature  shows  an  upward  tendency,  measurements  must  be 
continued  until  a  distinct  reaction  is  recognized  or  until  the  temperature 
begins  to  fall. 

"  (e)  If  a  reaction  is  detected  prior  to  the  sixteenth  hour,  the  measure- 
ments of  temperature  should  be  continued  until  the  expiration  of  this 
period. 

"(/)  If  there  is  an  unusual  change  of  temperature  of  the  stable,  or 
a  sudden  change  in  the  weather,  this  fact  should  be  recorded  on  the 
report  blank. 

"  (g)  If  a  cow  is  in  a  febrile  condition  tuberculin  should  not  be  used, 
because  it  would  be  impossible  to  determine  whether,  if  a  rise  of 
temperature  occurred,  it  was  due  to  the  tuberculin  or  to  some  transitory 
illness. 

"  (h)  Cows  should  not  be  tested  within  a  few  days  before  or  after 
calving,  for  experience  has  shown  that  the  result  at  these  times  may 
be  misleading. 

"  (i)  The  tuberculin  test  is  not  recommended  for  calves  under  three 
months  old. 

"  (j)  In  old,  emaciated  animals  and  in  re-tests,  use  twice  the  usual 
dose  of  tuberculin,  for  these  animals  are  less  sensitive. 

"  (k)  Condemned  cattle  must  be  removed  from  the  herd  and  kept 
away  from  those  that  are  healthy. 

"(/)  In  making  post-mortems  the  carcasses  should  be  thoroughly 
inspected,  and  all  of  the  organs  should  be  examined." 

Tuberculin  injections  are  also  made  in  man  to  reveal  a  suspected 
tuberculosis.  At  first  some  believed  that  the  irritation  aroused  in  the 
tuberculous  foci  by  the  tuberculin  sometimes  caused  a  dissemination  of 
the  bacilli  and  an  increase  in  the  disease.  When  carefully  used,  how- 
ever, in  suitable  cases  there  is  probably  no  danger.  A  drawback  to 
its  usefulness  is  that  it  does  not  reveal  the  extent  of  the  disease,  nor 
whether  the  tuberculosis  is  active.  It  is,  however,  of  great  value  in 
selected  cases,  both  surgical  and  medical,  where  slight  tuberculosis  is 
suspected,  and  yet  no  decision  can  be  reached.  In  the  small  doses 
advised  an  absolutely  latent  infection  would  probably  give  no  rise  of 
temperature.  I  quote  here  Dr.  Trudeau  upon  the  use  of  the  test: 


THE  BACILLUS  OF  TUBERCULOSIS  307 

"The  range  of  the  patient's  temperature  is  ascertained  by  taking  it 
at  8  A.M.,  3  P.M.,  and  8  P.M.  for  three  or  four  days  before  making  the 
test.  The  first  injection  should  not  exceed  0.5  nig.,  and  if  any  fever  is 
habitually  present  should  be  even  less,  and  is  best  given  early  in  the 
morning  or  late  at  night,  as  the  typical  reaction  usually  begins,  in  my 
experience,  within  six  or  twelve  hours.  Such  a  small  dose,  while  it  will 
often  be  sufficient  to  produce  the  looked-for  rise  of  temperature,  has, 
under  my  observation,  never  produced  unpleasant  or  violent  symp- 
toms. An  interval  of  two  or  three  days  should  be  allowed  between  each 
of  the  two  or  three  subsequent  injections  it  may  be  necessary  to  give, 
as  reaction  in  very  rare  cases  may  be  delayed  for  twenty-four  or  even 
thirty-six  hours.  On  the  third  day  a  second  dose  of  1  mg.  is  given,  and 
if  no  effect  is  produced  a  third,  of  2  mg.,  three  days  later.  In  the  great 
majority  of  cases  of  latent  tuberculosis  an  appreciable  reaction  will 
be  produced  by  the  time  a  dose  of  2  mg.  has  been  reached.  If  no  effect 
has  been  caused  by  the  tests  applied  as  above  I  have  usually  gone  no 
farther,  and  concluded  that  no  tuberculous  process  was  present,  or  at 
least  not  to  a  degree  which  need  be  taken  into  account  in  advising 
the  patient,  or  which  would  warrant  insisting  on  a  radical  change  in 
his  surroundings  and  mode  of  life.  If  some  slight  symptoms,  how- 
ever, have  been  produced  by  a  dose  of  2  mg.,  it  may  be  necessary 
to  give  a  fourth  injection  of  3  mg.  in  order  to  reach  a  positive  conclu- 
sion. Nevertheless,  it  should  be  borne  in  mind  that  in  a  few  cases  the 
exhibition  of  even  larger  doses  may  cause  reaction  and  indicate  the 
existence  of  some  slight  latent  tuberculous  lesion,  and  the  test  should 
not,  when  applied  within  the  moderate  doses  described,  be  considered 
absolutely  infallible. 

"No  evidence  in  connection  with  the  tuberculin  test  as  applied  to 
man  and  animals  has  been  forthcoming  thus  far  from  those  who  have 
made  use  of  it,  which  would  tend  to  sustain  the  general  impression 
that  this  method  is  necessarily  dangerous  and  tends  invariably  to  aggra- 
vate the  disease,  and  my  own  experience  has  developed  nothing  which 
would  seem  to  confirm  this  impression.  It  is  evident  that  the  size  of 
the  doses  given  has  much  to  do  with  the  limitations  of  this  method 
for  usefulness,  and  the  correctness  of  the  conclusions  reached  by  its 
application.  The  tuberculin  used  is  also  a  matter  of  some  importance 
in  determining  the  dosage,  as  different  samples  vary  considerably  in 
their  efficiency.  If  the  test  be  pushed  to  the  injection  of  such  large 
amounts  as  10  mg.  or  more,  as  advocated  by  Maragliano,  such  doses 
are  by  no  means  free  from  the  objection  of  occasionally  causing  un- 
pleasant and  sometimes  dangerous  symptoms;  and  even  if  the  amount 
given  be  not  carried  to  the  dose  of  10  mg.,  which  is  known  to  produce 
fever  in  healthy  subjects,  it  is  likely  that  on  account  of  individual  sus- 
ceptibility or  the  presence  of  some  other  morbid  process  in  the  body, 
reaction  will  be  found  to  occur  with  the  larger  doses  when  no  tubercu- 
lous process  exists.  The  adoption  of  an  initial  dose  so  small  as  to 
guard  against  the  absolute  possibility  of  producing  violent  reactionary 
symptoms,  and  the  graded  increase  of  the  subsequent  doses  within 


308  BACTERIA  PATHOGENIC  TO  MAN 

such  quantities  as  are  known  never  to  produce  reaction  in  healthy 
individuals,  would  seem  to  afford  the  best  protection  against  unpleasant 
results  and  misleading  evidence." 

Antituberculous  Serum. — Whether  serum  therapy  is  destined  to  solve 
the  problem  of  the  treatment  of  tuberculosis  remains  for  the  future  to 
decide,  but  up  to  the  present  the  results  obtained  with  antituberculous 
serum  do  not  warrant  our  forming  such  an  opinion.  Every  conceivable 
way  of  obtaining  the  true  products  of  the  tubercle  bacilli  has  been 
tried,  so  as  to  cause  the  injected  animals  to  produce  antibodies  both 
antitoxic  and  bactericidal.  At  present  Maragliano  and  Marmorek 
are  presenting  claims  that  their  sera  are  truly  curative.  Although 
both  these  men  have  had  a  large  experience  in  this  field  of  investigation, 
it  is  probable  that  the  final  judgment  will  be  that  little  good  comes 
from  the  injection  of  their  serum.  Very  few  observers  have  succeeded 
in  obtaining  appreciable  results  with  the  serums  prepared  by  other 
experimenters.  In  spite  of  much  conflicting  testimony,  it  is  probably 
safe  to  assert  that  no  sera  now  obtainable  have  any  great  value.  Nor 
as  we  look  at  the  progressive  nature  of  tuberculosis  can  we  see  much 
ground  to  hope  for  the  abundant  development  of  curative  substances 
in  the  blood  of  animals.  This  view,  however,  in  no  way  lessens  the 
necessity  of  continued  endeavor  until  every  method  conceivable  has 
ly^en  tried. 

Prophylaxis. — Meanwhile  all  energies  should  be  directed  to  the  pre- 
vention of  tuberculosis,  not  only  by  the  enforcement  of  proper  sanitary 
regulations  as  regards  the  care  of  sputum,  milk,  meat,  disinfection,  etc., 
but  also  by  continued  experimental  work  and  by  the  establishment  of 
free  consumptive  hospitals,  and  by  efforts  to  improve  the  character  of 
the  food,  dwellings,  and  condition  of  the  people  in  general,  we  should 
endeavor  to  build  up  the  individual  resistance  to  the  disease.  It  may 
be  years  yet  before  the  public  are  sufficiently  educated  to  co-operate 
with  the  sanitary  authorities  in  adopting  the  necessary  hygienic  meas- 
ures to  stamp  out  tuberculosis  entirely;  but,  judging  from  the  results 
-which  have  already  been  obtained  in  reducing  the  mortality  from  this 
dread  disease,  we  have  reason  to  believe  that  in  time  it  can  be  com- 
pletely controlled. 

The  Tubercle  Bacillus  of  Cattle  and  its  Relation  to  Human  Tuberculosis. 
— Among  the  domestic  animals  tuberculosis  is  most  common  in  cattle. 
On  account  of  the  milk  which  they  provide  for  our  use,  and  which  is 
liable  to  contain  bacilli,  the  relation  of  these  to  human  tuberculosis  is 
a  matter  of  extreme  importance. 

The  chief  seat  of  the  lesions  is  apt  to  be  the  lungs,  and  with  them  the 
pleura;  less  often  the  abdominal  organs  and  the  udder  are  affected. 
In  pigs  and  horses  the  abdominal  organs  are  most  often  involved,  then 
the  lungs  and  lymphatic  glands.  In  sheep  and  goats  tuberculosis  is 
rare.  The  bovine  bacillus,  as  the  most  important  of  the  group,  will  be 
alone  considered  here. 

The  bacilli  derived  from  cattle  are  on  the  average  a  little  shorter  and 
straighter  than  the  average  human  bacillus.  In  guinea-pigs,  and  espe- 


////:  />' ACILLUS  OF  TUBERCULOSIS  309 

cially  in  rabbits,  the  bovine  bacilii  are  more  virulent  than  the  majority 
of  those  from  human  sources.  Animals  infected  with  the  bacilli  from 
cattle,  as  well  as  those  from  the  other  domestic  animals,  react  to  the 
tuberculin  test.  All  these  bacilli  are,  therefore,  undoubtedly  from  the 
same  original  stock,  and  at  first  glance  we  might  consider  it  unnecessary 
to  prove  that  those  derived  from  cattle  were  capable  of  causing  human 
tuberculosis.  There  are  facts,  however,  which  have  made  this  inves- 
tigation of  great  importance.  As  we  investigate  we  find  that  all  facts 
tend  to  show  that  the  great  majority  of  cases  of  tuberculosis  in  human 
adults  come  from  human  infection.  The  cases  where  fairly  strong 
proof  of  bovine  infection  has  been  obtained  are  certainly  rare. 

Further,  we  have  the  undoubted  fact  that  the  long  sojourn  of  bacteria 
in  one  species  of  animal  tends  to  increase  the  virulence  of  the  germ 
for  that  animal  and  to  lessen  it  for  others. 

Theobald  Smith  has  made  the  interesting  discovery  that  there  is 
a  wide  difference  between  the  culture  growth  of  the  average  bovine 
bacillus  and  the  average  one  from  human  sources;  the  bovine  bacilli 
when  grown  in  glycerin  broth  causes  the  broth  to  become  less  and  less 
acid  and  finally  feebly  alkaline  to  phenolphthalein,  while  the  human 
types  cause  it  to  become  only  a  little  less  acid,  but  never  alkaline.  In 
ordinary  peptone  bouillon  the  reaction  cure  is  the  same  for  both. 
The  broth  becomes  alkaline.  Tuberculin  made  from  bovine  cultures 
is  alkaline  while  that  made  from  human  cultures  is  markedly  acid. 
Both  cultures  act  upon  the  glycerin,  but  in  different  ways.  He  had 
previously  noted  that  the  bovine  bacilli  in  cultures  were  shorter  and 
straighter,  and  grew  less  luxuriantly  than  those  from  man,  and,  further, 
that  the  bovine  bacilli  were  much  more  virulent  for  rabbits.  He  has 
found  these  differences  persist  for  long  periods,  and  believes  that  the 
simple  passage  through  a  single  person  in  a  case  of  human  tuberculosis 
would  not  be  sufficient  to  change  these  characteristics.  He  has  had  a 
chance  to  examine  the  bacilli  of  two  cases  in  young  children  which  were 
of  the  bovine  type,  but  in  adults  not  one  of  some  half  a  dozen  cultures 
showed  the  bovine  characteristics.  The  proof  that  Theobald  Smith 
gathered,  which  proved  the  difference  between  the  bovine  and  human 
bacilli,  has  enabled  him  and  others  to  conclusively  prove  that  a  con- 
siderable proportion  of  children  suffering  from  intestinal  or  mesen- 
teric  tuberculosis  aie  really  infected  with  bovine  bacilli.  These  bacilli 
are  capable  of  infecting  cattle. 

At  present  then  we  must  assume  that  bovine  bacilli  are  capable  of 
infecting  those  who  are  very  susceptible,  such  as  young  children. 
Whether  adults  are  also  infected  has  not  yet  been  decided.  Such  views 
as  Behring's,  that  much  adult  tuberculosis  is  due  to  infection  in  child- 
hood with  bovine  bacilli  which  have  remained  latent,  are  probably  very 
extreme.  This  question  is  in  great  need  of  further  study.  At  present 
no  cattle  which  are  tuberculous  should  be  allowed  to  furnish  milk,  or 
at  least  such  milk  should  not  be  used  for  drinking  purposes  without 
being  sterilized.  The  flesh  is  less  harmful,  as  muscular  tissue  is  seldom 
infected. 


310  BACTERIA  PATHOGENIC  TO  MAN 

Bird  (Avian)  Tuberculosis. — Tuberculosis  is  very  common  and  infec- 
tious among  fowls.  The  bacilli  themselves  grow  more  readily  on 
artificial  culture  media  and  produce  a  more  even  and  moist  growth. 
They  are  able  to  develop  at  a  temperature  of  43.5°  C.,  which  is  above 
that  at  which  the  human  and  bovine  types  can  usually  grow.  The 
bacilli  are  more  apt  to  show  branching  forms  than  the  human.  In 
rabbits  they  produce  very  similar  lesions,  but  guinea-pigs  are  much 
less  susceptible.  Birds  are  much  less  susceptible  to  bacilli  from  human 
or  bovine  sources  than  to  those  from  birds.  Nocard  states  that  bacilli 
from  human  sources  placed  in  collodion  sacs  and  inserted  into  the  peri- 
toneal cavity  of  birds  gradually  acquire  avarian  characteristics,  so  that 
after  eighteen  months  they  can  readily  infect  fowls  and  approach  the 
avian  cultural  type.  This  suggests  that  bovine  bacilli  remaining  in 
man  for  years  might  acquire  human-type  characteristics.  They  are 
undoubtedly  from  the  same  stock  as  the  mammalian  varieties,  but  have 
become  modified;  it  is  not  believed  that  they  are  any  factor  in  the 
production  of  human  tuberculosis. 

Tuberculosis  in  Fish. — In  certain  species  of  fish  a  tuberculous  disease 
has  been  noted.  The  bacilli  have  the  staining  characteristics  of  the 
warm-blooded  types,  but  do  not  grow  at  body  temperature  and  do 
not  affect  mammals. 

Methods  of  Examination  for  Tubercle  Bacilli  and  Other  Associated 

Bacteria. 

One  of  the  most  important  results  of  the  discovery  of  the  tubercle 
bacillus  relates  to  the  practical  diagnosis  of  tuberculosis.  The  staining 
peculiarities  of  this  bacillus  renders  it  possible  by  the  bacteriological 
examination  of  microscopic  preparations  to  make  an  almost  abso- 
lutely positive  diagnosis  in  the  majority  of  cases.  A  still  more  certain 
test  in  doubtful  cases  is  the  subcutaneous  or  intraperitoneal  injection 
of  guinea-pigs,  which  permits  of  the  determination  of  the  presence  of 
numbers  of  bacilli,  so  small  as  to  escape  detection  by  microscopic 
examination.  For  the  animal  test,  however,  time  is  required — at  least 
three  weeks,  and,  when  the  result  is  negative,  at  least  six  weeks — before 
any  positive  conclusion  can  be  reached,  for  when  only  a  few  bacilli  are 
present  tuberculosis  develops  slowly  in  animals. 

Microscopic  Examination  of  Sputum  for  the  Presence  of  Tubercle  Bacilli. 
1.  COLLECTION  OF  MATERIAL. — The  sputum  should  be  collected  in  a 
clean  bottle  (two  ounce)  with  a  wide  mouth  and  a  water-tight  stopper, 
and  the  bottle  labelled  with  the  name  of  the  patient  or  with  some  other 
distinguishing  mark.  The  expectoration  discharged  in  the  morning  is 
to  be  preferred,  especially  in  recent  cases,  and  the  material  should  be 
coughed  up  from  the  lungs.  Care  should  be  taken  that  the  contents 
of  the  stomach,  nasopharyngeal  mucus,  etc.,  are  not  discharged  during 
the  act  of  expectoration  and  collected  instead  of  pulmonary  sputum. 
If  the  expectoration  be  scanty  the  entire  amount  discharged  in  twenty- 
four  hours  should  be  collected.  In  pulmonary  tuberculosis  the  puru- 


THE  BACILLUS  OF  TUBERCULOSIS  311 

lent,  cheesy,  and  mucopurulent  sputum  usually  contains  bacilli;  while 
pure  mucus,  blood,  and  saliva,  as  a  rule,  do  not.  When  hemorrhage 
has  occurred,  if  possible,  some  purulent,  cheesy,  or  mucopurulent  sputum 
should  be  collected  for  examination.  The  sputum  should  not  be  kept 
any  longer  than  necessary  before  examination,  for,  though  a  slight 
delay  or  even  till  putrefaction  begins,  does  not  vitiate  the  result  so  far 
as  the  examination  for  tubercle  bacilli  is  concerned,  it  almost  destroys 
any  proper  investigation  of  the  mixed  infection  present;  it  is  best,  there- 
fore, to  examine  it  in  as  fresh  a  condition  as  possible,  and  it  should  be 
kept  on  ice  until  examined  if  cultures  are  to  be  made. 

2.  METHODS  OF  EXAMINATION.  Examination  for  Tubercle  Bacilli. 
— Pour  the  specimen  into  a  clean,  shallow  vessel,  having  a  blackened 
bottom — a  Petri  dish  placed  upon  a  sheet  of  dull  black  paper  answers 
the  purpose — and  select  from  the  sputum  some  of  the  true  expectora- 
tion, containing,  if  possible,  one  of  the  small  white  or  yellowish-white 
cheesy  masses  or  "  balls"  which  it  contains.  From  this  make  rather 
thick  cover-glass  or  slide  "smears"  in  the  usual  way.  In  doubtful 
cases  a  number  of  these  coarse  or  fine  particles  should  be  placed  on 
the  slide.  The  material  being  thick,  should  be  evenly  spread  and  very 
thoroughly  dried  in  the  air  before  heating.  Immerse  this  in  a  solution  of 
Ehrlich's  aniline-water  fuchsin,  contained  in  a  thin  watch-glass  or  porce- 
lain dish,  and  steam  over  a  small  flame  for  two  minutes.  Then  remove 
the  glass  from  this  and  wash  with  water.  Now  decolorize  by  immersing 
the  stained  preparation  in  a  3  per  cent,  hydrochloric  acid  solution  in 
alcohol  for  from  one-half  up  to  one  minute,  removing  at  the  time  when 
all  color  is  just  about  gone  from  the  cover-glass  smear.  Wash  thor- 
oughly with  water,  and  make  a  contrast  stain  by  applying  a  cold  solu- 
tion of  Loeffler's  alkaline  methylene  blue- 
Concentrated  alcoholic  solution  of  methylene  blue  .  .  30  c.c. 
Caustic  potash  (1:10,000  solution) 100  " 

for  from  fifteen  to  thirty  seconds.  Wash  with  water;  press  between 
folds  of  filter  paper;  dry  in  the  air;  mount  and  examine. 

The  tubercle  bacilli  are  distinguished  by  the  fact  that  they  retain 
the  red  color  imparted  to  them  in  the  fuchsin  solution,  while  the  other 
bacteria  present,  having  been  decolorized  in  the  acid  solution,  take 
the  contrast  stain  and  appear  blue.  (See  Plate  L,  Figs.  1  and  2.) 

Various  methods  have  been  suggested  for  the  staining  of  tubercle 
bacilli,  but  the  original  method  as  employed  by  Koch,  or  some  slight 
modification  of  it,  is  so  satisfactory  in  its  results  that  it  seems  unneces- 
sary to  substitute  others  for  it.  The  above  is  a  slight  modification  of 
the  Koch-Ehrlich  method,  differing  from  it  chiefly  in  the  use  of  a  weak 
for  a  strong  acid  decolorizer.  It  has  been  found  that  the  strong  acid 
solution  originally  employed  (5  per  cent,  sulphuric  acid  solution  in 
alcohol)  often  decolorizes  some  of  the  bacilli  entirely  by  its  too  energetic 
action,  and  that  a  weaker  decolorizer,  such  as  the  above,  gives  more 
uniform  results. 


312  BACTERIA  PATHOGENIC  TO  MAN 

Instead  of  the  Koch-Ehrlich  aniline-water  solution,  Ziehl's  carbol- 
fuchsin  solution  may  be  used,  and  is  by  many  preferred.  Instead  of 
floating  the  cover-glass  smears  on  the  staining  fluid  they  can  be  held 
in  the  Cornet  forceps,  covered  and  kept  covered  completely  with  fluid 
while  steamed  for  two  minutes  over  the  flame. 

The  Koch-Ehrlich  aniline-water  solution  decomposes  after  having 
been  made  for  a  time,  so  that  it  must  be  freshly  prepared  as  needed. 
Solutions  older  than  fourteen  days  should  not  be  used.  The  advan- 
tages in  using  Ziehl's  carbol-fuchsin  solution  are  that  it  keeps  well 
and  is  more  convenient  for  use  in  small  quantities. 

Another  method,  which  is  often  of  value  on  account  of  its  simplicity 
and  rapidity  of  performance,  is  that  of  Frankel  as  modified  by  Gab- 
bett.  This  consists  in  staining  the  cover-glass  "smear"  with  steaming 
ZiehPs  carbol-fuchsin  solution  for  from  one  to  two  minutes,  and  then, 
after  washing  in  water,  placing  it  from  one-half  to  one  minute  directly 
in  a  second  solution  which  contains  both  the  acid  for  decolorizing  and 
the  contrast  stain.  This  second  solution  consists  of— 

Sulphuric  acid 25  c.c. 

Methylene  blue  in  substance         ......       2  grm. 

Water 75  c.c. 

It  is  then  washed  with  water  and  is  ready  for  examination.  The  tubercle 
bacilli  will  remain  red  as  stained  by  the  fuchsin,  while  all  the  other 
bacteria  will  be  tinted  blue. 

When  the  number  of  tubercle  bacilli  in  sputum  is  very  small  they 
may  easily  escape  detection.  Methods  have,  therefore,  been  suggested 
for  finding  them  under  these  circumstances.  Ribbert  proposed  the 
addition  to  the  sputum  of  an  equal  amount  or  more  of  a  2  per  cent,  solu- 
tion of  caustic  potash,  and  boiling  the  mixture.  The  mucus  is  stirred 
slightly  until  it  is  dissolved.  To  this  an  equal  amount  of  water  is  stirred 
in  and  the  whole  is  placed  in  a  conical  glass  vessel;  and  any  bacilli  present 
are  deposited  at  the  bottom  and  may  be  found  in  the  sediment  after 
removing  the  supernatant  fluid.  The  sedimentation  may  be  obtained 
more  quickly  by  the  centrifugal  machine. 

Detection  of  Tubercle  Bacilli  in  Urine  and  Feces. — The  catheterized 
urine  is  centrifuged.  If  little  sediment  appears,  the  upper  portion  of  the 
fluid  is  removed  and  the  remainder  again  centrifuged.  If  the  urine  is 
rich  in  salts  of  uric  acid,  the  same  may  be  diminished  by  carefully  warm- 
ing the  urine  before  treating  it.  If  too  alkaline  add  a  little  acetic  acid. 

The  feces  are  examined  for  any  purulent  or  mucous  particles.  If 
none  are  found  the  larger  masses  of  feces  are  removed  and  then  the  rest 
diluted  and  centrifugalized.  The  examiner  must  remember  that  bacilli 
swallowed  with  the  sputum  may  appear  in  the  feces. 

Examination  for  Other  Bacteria  (Mixed  Infection). — With  regard  to 
the  bacteriological  diagnosis  of  pulmonary  phthisis,  many  consider  that 
it  is  not  enough  to  show  only  the  presence  of  tubercle  bacilli;  it  is  held 
to  be  of  importance,  both  for  purposes  of  prognosis  and  treatment, 
that  the  presence  of  other  micro-organisms  which  may  be  associated 


THE  BACILLUS  OF  TUBERCULOSIS 

with  the  tubercle  bacillus  should,  also  be  determined.  It  is  now  usual 
to  distinguish  pure  tuberculosis  of  the  lungs  from  a  mixed  infection. 
Phthisis  due  to  the  tubercle  bacillus  alone,  which  constitutes  but  a 
small  percentage  of  all  cases,  may  occur  almost  without  febrile  reac- 
tion; or  when  fever  occurs  the  prognosis  is  unfavorable,  thus  indicating 
that  the  disease  is  already  advanced.  It  is  in  the  uncomplicated  forms 
of  phthisis,  moreover,  where  one  must  expect  if  anywhere  the  best  results 
from  treatment  with  tuberculin  or  antituberculous  serum.  The  majority 
of  cases,  however,  of  pulmonary  tuberculosis  show  a  mixed  infection, 
especially  with  varieties  of  the  streptococcus  and  pneumococcus.  These 
cases  may  be  active,  with  fever,  or  passive,  without  fever,  according, 
perhaps,  as  the  parenchyma  of  the  lung  is  invaded  by  the  bacteria;  or 
they  are  only  superficially  located  in  cavities,  bronchi,  etc.  Mixed 
infection  with  the  staphylococcus,  other  micrococci,  and  with  the  influ- 
enza bacilli  have  also  been  frequently  met  with  by  us.  The  tetragenus 
has  not  been  often  detected  by  us  in  thoroughly  washed  fresh  sputum. 
At  present  the  facts  seem  to  prove  that  the  tubercle  bacilli  have  in  the 
great  majority  of  cases,  at  least  until  shortly  before  death,  a  much  more 
important  role  than  the  associated  bacteria. 

Sputum  Washing. — Some  of  the  associated  bacteria  found  in  the 
expectoration  come  from  the  diseased  areas  of  the  lungs,  while  others 
were  merely  added  to  the  sputa  as  it  passed  through  the  mouth,  or 
have  developed  after  gathering.  To  endeavor  to  separate  the  one 
from  the  other  we  wash  the  sputa.  The  first  essential  is  that  the  mate- 
rial be  washed  within  a  few  minutes,  and  certainly  within  an  hour,  of 
being  expectorated.  If  a  longer  time  is  allowed  to  intervene,  the  bac- 
teria from  the  mouth  will  penetrate  into  the  interior  of  the  mucus,  and 
thus  appear  as  if  they  came  from  the  lungs.  Sputum  treated  twenty- 
four  hours  after  its  expectoration  is  useless  for  examining  for  anything 
except  the  tubercle  bacillus.  A  rough  method  is  to  pour  some  of  the 
specimen  of  sputum  to  be  examined  into  a  convenient  receptacle  con- 
taining sterile  water,  and  withdraw,  by  means  of  a  sterilized  platinum 
wire,  one  of  the  cheesy  masses  or  thick  "balls"  of  mucus.  Pass  this 
loop  five  times  through  sterile  water  in  a  dish;  repeat  the  operation  in 
fresh  water  in  a  second  and  third  dish.  Spread  what  remains  of  the 
mass  on  cover-glasses  and  make  smear  preparations;  stain  and  examine. 
With  another  mass  inoculate  ascitic  bouillon  in  tubes  and  agar  in  plates. 

When  we  wish  to  thoroughly  exclude  mouth  bacteria,  a  lump  of  the 
sputum  raised  by  a  natural  cough  is  seized  by  the  forceps  and  trans- 
ferred to  a  bottle  of  sterile  water  and  thoroughly  shaken;  it  is  then 
removed  to  a  second  bottle  of  bouillon  and  again  thoroughly  shaken. 
From  this  it  is  passed  in  the  same  way  through  four  other  bottles  of 
bouillon.  A  portion  of  the  mass  is  now  smeared  over  cover-glasses, 
and  the  rest  inoculated  in  suitable  media,  such  as  agar  in  Petrie  dishes, 
and  ascitic  fluid  bouillon  in  tubes.  If  desired  the  bacteria  washed  off 
in  the  different  washings  are  allowed  to  develop. 

Practical  Notes  on  the  Examination  for  Mixed  Infection. — 1.  The 
difficulties  to  be  overcome,  in  order  to  obtain  sputum  consisting  pre- 


314  BACTERIA  PATHOGENIC  TO  MAN 

sumably  of  exudate  from  the  deeper  portions  of  the  lungs,  are  so  great 
that  the  collection  of  the  specimens  should  be  supervised  by  the  bacteri- 
ologist in  charge  of  the  work  of  examination. 

2.  Specimens  of  sputum  collected  even  with  the  greatest  precaution 
may  give   evidence   of  decided   mouth   infection   unless   immediately 
washed. 

3.  The  sputum  must  be  examined  very  soon  after  collection. 

4.  The  culture  medium  used  for  the  final  cultures  must  be  suitable 
for  the  growth  of  the  micro-organisms. 

5.  At  least  two  successive  examinations  of  sputum  should  be  made 
in  each  case. 

6.  The  results,  especially  as  to  the  number  of  colonies,  vary  accord- 
ing to  the  size  and  tenacity  of  the  ball  of  sputum  washed — e.  g.,  a  small 
ball  of  sputum  which  becomes  more  or  less  broken  up  upon  thorough 
shaking  may  contain  very  few  or  no  bacteria. 

Williams,  in  the  examination  of  the  sputum  in  some  40  cases,  came 
to  the  following  conclusions:  1.  The  presence  of  a  large  number  of 
bacteria  in  a  satisfactory  and  thoroughly  washed  specimen  of  sputum 
indicates  that  these  bacteria  probably  play  an  active  part  in  the  disease. 

2.  The  presence  of  a  small  number  of  bacteria  in  such  sputum  does 
not  necessarily  indicate  that  they  are  not  active  in  that  case,  for  they 
may  penetrate  more  or  less  deeply  into  the  lung  tissue,  and  produce 
pathological  changes  without  being  thrown  off  in  large  numbers  with 
the  exudate.    It  is  probable,  however,  that,  as  a  rule,  the  smaller  the 
number  found  the  less  the  degree  of  mixed  infection. 

3.  Cases  of  clinically  secondary  infection  frequently  give  pure  cul- 
tures of  some  one  organism,  which  appear  to  be  capable  of  causing  the 
symptoms. 

4.  In  the  majority  of  severe  cases  of  clinically  mixed  infection  many 
organisms  have  been  found  which  usually  have  belonged  to  several 
different  species  or  varieties. 

5.  In  the  majority  of  cases  of  clinically  non-mixed  infection  very 
few  organisms  have  been  found. 

6.  Only    bacteria    which    might    cause    pathological    changes  were 
present. 

7.  Very  few  of  the  organisms  found  were  very  virulent  in  rabbits, 
even  though  coming  from  severe  cases  of  mixed  infection. 

The  virulence  for  laboratory  animals  of  bacteria  obtained  from  the 
sputum  is,  therefore,  no  indication  of  their  virulence  for  man,  because 
of  the  impossibility  of  reproducing  in  such  animals  the  exact  condition 
of  susceptibility  present  in  human  infection. 

General  Rules  in  Microscopic  Examination  of  Sputum. — Always  make 
two  still  preparations  from  each  specimen.  Report  no  result  as 
negative  until  at  least  two  preparations  have  been  subjected  to  a 
thorough  search  with  a  1/12  oil-immersion  or  2  mm.  apochromatic  lens 
by  means  of  a  mechanical  stage.  From  a  very  large  experience  in 
the  examination  of  sputum  for  tubercle  bacilli,  the  New  York  Health 
Department  bacteriologists  have  concluded  that  the  examination  of 


Till-:  HACILLUS  OF  TUBERCULOSIS  315 

two  preparations  of  each  specimen,  in  the  careful  manner  described 
above,  is  usually  sufficient  to  demonstrate  the  presence  of  the  bacilli 
when  they  are  present  in  the  sputa,  and  they  are  usually  found  to  be 
present  to  this  extent  in  fairly  well-developed  cases  of  pulmonary 
tuberculosis,  and  in  many  cases  which  are  in  the  incipient  stage.  There 
are,  however,  undoubted  cases  of  incipient  pulmonary  tuberculosis 
which  require  the  examination  of  many  preparations  before  the 
tubercle  bacillus  can  be  found;  and  cases  also  occur  in  which  the  sputum 
for  a  time  does  not  contain  the  bacilli,  which  were,  nevertheless, 
present  at  an  earlier  period,  and  which  again  later  appear.  Therefore, 
if  cases  occur  which  may  be  still  regarded  as  possibly  tuberculosis, 
further  examinations  of  the  sputum  should  be  made.  It  should  also 
be  constantly  borne  in  mind  that  the  demonstration  of  the  presence 
of  tubercle  bacilli  in  the  sputum  proves  about  as  conclusively  as  any- 
thing can  the  existence  of  some  degree  of  tuberculosis;  but  that  the 
absence  of  tubercle  bacilli  or  the  failure  to  find  them  microscopically 
does  not  positively  exclude  the  existence  of  the  disease.  Here  tuberculin 
can  be  made  use  of. 

Staining  of  Tubercle  Bacilli  in  Tissues. — Thin  sections  of  tuberculous 
tissues  may  be  stained  by  the  same  methods  recommended  for  cover- 
glass  preparations,  except  that  it  is  best  not  to  employ  heat  to  any 
•extent.  Fixation  in  bichloride  of  mercury  is  better  than  in  alcohol. 
Formalin  is  a  bad  fixative,  as  it  makes  the  tissues  hold  the  fuchsin  with 
as  much  tenacity  as  the  bacilli.  Both  paraffin  and  celloidin  may  be 
used  for  embedding,  but  the  former  is  better. 

EHRLICH'S  METHOD. — Place  the  paraffin  sections  in  aniline  fuchsin 
and  leave  at  37°  C.  for  from  six  to  twelve  hours,  or  at  about  80°  C.  for 
three  to  five  minutes,  the  sections  are  then  washed  in  water;  then 
decolorize  by  placing  them  for  about  half  a  minute  in  dilute  nitric  acid 
(10  per  cent.),  or  in  3  per  cent,  hydrochloric  acid  in  alcohol;  wash  in 
60  per  cent,  alcohol  until  no  more  color  is  given  off;  counterstain  for 
two  or  three  minutes  in  a  saturated  aqueous  solution  of  methylene  blue, 
or,  better,  with  haematoxylin;  wash  in  water;  dehydrate  with  absolute 
alcohol;  clear  in  oil  of  cedar  or  xylol,  and  mount  in  xylol  balsam. 

METHOD  OF  ZIEHL-NEELSON. — Stain  the  section  in  warmed  carbol- 
fuchsin  solution  for  one  hour;  the  temperature  to  be  not  over  45°  to 
50°  C.  Decolorize  for  a  few  seconds  in  5  per  cent,  sulphuric  acid, 
then  in  70  per  cent,  alcohol,  and  from  this  on  as  in  the  Ehrlich  method. 

Inoculation  of  Animals. — The  inoculation  of  suspected  material  into 
guinea-pigs  sometimes  produces  tuberculosis  when  no  bacilli  could  be 
detected  by  microscopic  examination.  The  material  may  be  injected 
into  the  subcutaneous  tissues,  into  the  peritoneal  cavity,  or  into  the 
mammary  gland  of  a  pregnant  guinea-pig. 

Cultivation. — This  is  so  difficult  and  requires  so  much  time  that  it  is 
not  used  except  in  important  investigations  upon  the  nature  of  the 
tubercle  bacilli. 


CHAPTER   XXII. 

BACILLI  SHOWING  STAINING  REACTIONS  SIMILAR  TO  THOSE  OF 

THE  TUBERCLE  BACILLI— LUSTGARTEN'S  BACILLUS— SMEGMA 

BACILLUS— LEPROSY  BACILLUS— GRASS  BACILLI. 

Lust gart en's  Bacillus — Smegma  Bacillus. 

BACILLI  were  discovered  by  Lustgarten  in  syphilitic  lesions  of 
syphilitic  ulcers  (1884),  and  believed  by  him  to  be  the  specific  cause 
of  this  disease.  It  has  since  been  shown  that  in  normal  smegma  from 
the  prepuce  or  the  vulva  bacilli  are  found  in  great  abundance,  similar 
in  their  morphology  to  the  bacillus  of  Lustgarten,  but  differing,  as  a 
rule,  slightly  in  certain  staining  peculiarities.  (See  Fig.  98.) 

Morphology. — Straight  or  curved  bacilli,  which  bear  considerable 
resemblance  to  tubercle  bacilli,  but  differ  from  them  in  staining  reactions. 

The  bacilli  are  not  usually  found  free  in  the 
tissues,  but  commonly  lie  singly  or  some- 
times in  groups  within  the  interior  of  cells, 
having  a  round,  oval,  or  polygonal  form, 
and  apparently  somewhat  swollen. 

Staining.  —  The    bacillus    of    Lustgarten 
stains  with  almost  as  much  difficulty  as  the 
tubercle  bacillus,  but  is  much  less  resistant 
^  ^  ^  to  the  action  of  certain  decolorizing  agents, 

such  as  mineral  acids,  particularly  sulphuric 
acid. 

Biological    and    Pathogenic    Properties.— 
smegma  bacilli,  similar  in  char-  Numerous  attempts  have  been  made  to  cul- 

acteristics  to  Lustgarten's  bacillus.     ,.  .1         i        *n  p     T  *• 

x  1100  diameters.  tivate  the    bacillus  of    Lustgarten  on   arti- 

ficial   media,    but    with    doubtful    success. 
The  inoculation  of  animals  has  also  given  only  negative  results. 

Lustgarten's  bacillus  has  been  found  in  various  syphilitic  tissues  and 
lesions,  in  beginning  sclerosis,  in  the  papules,  in  condylomata  and 
gummata,  and  not  only  in  the  vicinity  of  the  genitals,  but  also  in  the 
mouth,  throat,  heart,  and  brain.  No  satisfactory  experimental  evidence 
has  been  given  of  its  causative  relation  to  syphilis.  The  finding  of 
saprophytic  bacilli — the  so-called  smegma  bacilli — (Fig.  98  and  Plate  L, 
Fig.  4)  almost  identical  morphologically  with  the  bacillus  of  Lustgarten, 
under  the  prepuce  of  healthy  persons,  does  not  prove  the  identity  of 
the  two  bacilli,  though,  in  the  absence  of  cultures  and  inoculation  experi- 
ments, we  have  not  the  means  of  establishing  their  relationship  to  one 
another.  The  smegma  bacilli  have  never  been  identified  in  other  parts- 


BACILLI  RESEMBLING  TUBERCLE  BACILLI  IN  STAINING    317 

of  the  body,  except  in  the  neighborhood  of  the  genitals.  While  the 
bacillus  of  Lustgarten  cannot  resist  the  prolonged  decolorizing  action 
of  acids,  but  is  resistant  to  the  action  of  alcohol,  the  smegma  bacillus, 
when  stained,  is  quickly  decolorized  by  alcohol,  but  quite  resistant  to 
5  per  cent,  sulphuric  acid  solution.  Besides,  the  syphilis  bacillus  has 
been  found  in  papules,  in  gummata,  and  other  syphilomata,  where  there 
seems  no  probability  whatever  of  the  smegma  bacillus  having  emigrated. 
Finally,  other  bacilli  have  been  described  and  claimed  to  be  the  specific 
cause  of  syphilis,  but  none  of  these  discoveries  have  been  confirmed. 
The  latest  micro-organism  is  discussed  under  protozoa. 

Syphilitic  Infection. — Infection  of  those  not  immune  can  take  place 
at  any  time  when  an  abrasion,  however  small,  is  brought  in  contact 
with  the  blood  or  secretions  from  the  primary  or  secondary  lesions  of 
syphilitics. 

The  differential  diagnosis  of  Lustgarten's  bacillus  must  be  made 
from  the  tubercle  bacillus,  the  smegma  bacillus,  and  the  leprosy  bacillus. 
According  to  Hueppe,  the  differential  diagnosis  between  these  four 
organisms  depends  upon  the  following  reactions:  When  stained  by 
the  carbol-fuchsin  method,  commonly  em  ployed  in  staining  the  tubercle 
bacillus,  the  syphilis  bacillus  becomes  almost  instantly  decolorized  by 
treatment  with  mineral  acids,  particularly  sulphuric  acid;  whereas,  the 
smegma  bacillus  resists  such  treatment  for  a  much  longer  time,  and 
the  lepra  and  tubercle  bacillus  for  a  still  longer  time.  On  the  other 
hand,  if  decolorization  is  practised  with  alcohol  instead  of  acids,  the 
smegma  bacillus  is  the  first  to  lose  its  color.  The  bacillus  tuberculosis 
and  the  bacillus  of  leprosy  are  both  very  retentive  of  their  color,  even 
after  treatment  with  acids  and  alcohol.  If,  then,  we  treat  the  preparation, 
stained  with  carbol  fuchsin,  with  sulphuric  acid,  the  syphilis  bacillus 
becomes  almost  at  once  decolorized.  If  it  is  not  immediately  decolorized, 
treat  with  alcohol;  if  it  is  then  decolorized,  it  is  the  smegma  bacillus. 
If  it  is  still  not  decolorized,  it  is  either  the  leprosy  or  the  tubercle  bacillus. 

By  these  methods  the  differential  diagnosis  can  usually  be  made. 
In  all  investigations  of  importance,  however,  animal  inoculations  should 
also  be  made,  as  by  this  means  alone  can  a  positive  diagnosis  from 
tuberculosis  be  established.  Especial  care  should  be  observed  in  the 
examination  of  syphilitic  ulcers  of  the  genital  region,  as  in  this  situation 
the  smegma  bacilli  are  almost  always  present. 

Leprosy  Bacillus. 

The  bacillus  of  leprosy  was  discovered  by  Hansen  and  Neisser  (1879) 
in  the  leprous  tubercles  of  persons  afflicted  with  the  disease.  This 
discovery  was  confirmed  by  many  subsequent  observers. 

Morphology. — Small,  slender  rods  resembling  the  tubercle  bacilli 
in  form,  but  somewhat  shorter  and  not  so  frequently  curved.  The 
rods  have  pointed  ends,  and  in  stained  preparations  unstained  spaces, 
similar  to  those  observed  in  the  tubercle  bacillus,  are  seen.  They  stain 
readily  with  the  aniline  colors  and  also  by  Gram's  method.  Although 


318  BACTERIA  PATHOGENIC  TO  MAN 

differing  slightly  from  the  tubercle  bacillus  in  the  ease  with  which  they 
take  up  the  ordinary  aniline  dyes,  they  behave  like  tubercle  bacilli  in 
retaining  their  color  when  subsequently  treated  with  strong  solutions 
of  the  mineral  acids  and  alcohol.  The  slight  difference  in  staining 
characteristics  is  too  little  to  be  relied  upon  for  diagnostic  purposes. 

Biological  Characters. — Attempts  to  cultivate  the  bacillus  leprse  have 
been  frequently  made,  but  so  far  with  negative  results. 

Pathogenesis. — Numerous  inoculation  experiments  have  been  made 
on  animals  with  portions  of  leprous  tubercles,  but  there  is  no  conclusive 
evidence  that  leprosy  can  be  transmitted  to  the  lower  animals  by  inocu- 
lation. The  inference  that  this  bacillus  bears  an  etiological  relation 
to  the  disease  with  which  it  is  associated  is  based  entirely  upon  the 
demonstration  of  its  constant  presence  in  leprous  tissues  (Fig.  99). 


FIG.  99 


Leprosy  bacilli  in  nodule.    (Kolle  and  Wassermann.) 

The  bacilli  are  found  in  all  the  diseased  parts,  and  usually  in  large 
numbers,  especially  in  tubercles  on  the  skin,  in  the  conjunctiva  and 
cornea,  the  mucous  membranes  of  the  mouth,  gums,  and  larynx, 
and  in  the  interstitial  processes  of  the  nerves,  testicles,  spleen,  liver, 
and  kidneys.  The  rods  lie  almost  exclusively  within  the  peculiar  round 
or  oval  cells  of  the  granulation  tissue  which  composes  the  leprous 
tubercles,  either  irregularly  scattered  or  arranged  parallel  to  one  another. 
In  old  centres  of  infection  the  leprosy  cells  containing  the  bacilli  are 
larger  and  often  polynuclear.  Giant  cells,  such  as  are  found  in  tuber- 
culosis, are  claimed  to  have  been  observed  by  a  few  investigators  (Boinet 
and  Borrel).  In  the  interior  of  the  skin  tubercles,  the  hair  follicles, 
sebaceous  and  sweat-glands  are  often  attacked,  and  bacilli  have  some- 
times been  found  in  these  (Unna,  etc.).  Quite  young  eruptions  often 
contain  a  few  bacilli.  A  true  caseation  of  the  tubercles  does  not  occur, 
but  ulceration  results. 

In  the  anaesthetic  forms  of  leprosy  the  bacilli  are  found  most  commonly 
in  the  nerves  and  less  frequently  in  the  skin.  They  have  been  demon- 


BACILLI  RESEMBLING  TUBERCLE  BACILLI  IN  STAINING     319 

strated  in  the  sympathetic  nervous  system,  in  the  spinal  cord,  and  in 
the  brain.  The  bacillus  lepne  occurs  also  in  the  blood,  partly  free  and 
partly  within  the  leukocytes,  especially  during  the  febrile  stage  which 
precedes  the  breaking  out  of  fresh  tubercles  (Walters  and  Doutrelepont). 
The  bacilli  have  also  been  found  in  the  intestines,  in  the  lungs,  and  in 
the  sputum,  but  not  in  the  urine. 

With  regard  to  the  question  of  the  direct  inheritance  of  the  disease 
from  the  mother  to  the  unborn  child  there  is  considerable  difference  of 
opinion.  Some  cases  have  been  reported,  however,  in  which  a  direct 
transmission  of  the  bacillus  during  intrauterine  life  seems  to  be  the 
only  or  most  plausible  explanation  of  the  infection.  At  the  same 
time,  we  have  no  positive  experimental  evidence  to  prove  that  such  an 
infection  does  take  place.  Although  many  attempts  have  been  made 
to  infect  healthy  individuals  with  material  containing  the  bacilli  of 
leprosy,  the  results  are  not  conclusive.  Even  the  experiments  made  by 
Arning,  wrho  successfully  infected  a  condemned  criminal  in  the  Sandwich 
Islands  with  fresh  leprous  tubercles,  and  which  has  been  regarded  as 
positive  evidence  of  the  transmissibility  of  the  disease  in  this  way,  is  by 
no  means  conclusive;  for,  according  to  Swift,  the  man  had  other  oppor- 
tunities for  becoming  infected.  These  negative  results,  together  with 
the  fact  that  infection  does  not  more  frequently  occur  in  persons  exposed 
to  the  disease,  may  possibly  be  explained  by  the  assumption  that  the 
bacilli  contained  in  the  tuberculous  tissue  are  mostly  dead,  or  much 
more  probably  that  an  individual  susceptibility  to  the  disease  is  requisite 
for  its  production. 

The  widespread  idea,  before  the  discovery  of  the  leprosy  bacillus, 
that  the  disease  was  associated  with  the  constant  eating  of  dried  fish 
or  a  certain  kind  of  food,  has  now  been  entirely  abandoned. 

The  relation  of  leprosy  to  tuberculosis  is  sufficiently  evident  from 
their  great  similarity  in  many  respects.  This  is  rendered  still  more 
remarkable  if  the  observation  recently  made  is  true,  that  leprosy  reacts, 
both  locally  and  generally,  to  an  injection  of  tuberculin  in  the  same 
manner  as  tuberculosis  (Babes  and  Kalindero). 

Differential  Diagnosis. — The  differential  diagnosis  between  leprosy 
and  tuberculosis  is  not  difficult  in  typical  cases.  The  large  numbers 
of  bacilli  found  in  the  interior  of  the  cells  would  point  with  great  prob- 
ability to  leprosy.  Too  much  importance  should  not  be  placed  upon 
the  staining  peculiarities,  as  these  are  not  constant.  Moreover,  the  two 
diseases  not  infrequently  occur  together  in  the  same  individual.  In 
making  the  diagnosis,  therefore,  all  the  signs,  histological  and  patho- 
genic, must  be  considered  and  animal  inoculations  made. 

Timothy  and  Other  Grass  Bacilli. 

On  various  grasses,  in  cows'  manure,  in  butter,  and  in  milk,  there 
have  been  discovered  a  number  of  varieties  of  bacteria  which  have 
more  or  less  of  the  characteristics  of  the  tubercle  bacillus.  Some  of 
them  are  as  difficult  to  stain  and  as  resistant  to  the  decolorizing  action  of 


320  BACTERIA  PATHOGENIC  IN  MAN 

mineral  acids  and  alcohol  as  the  tubercle  bacillus  found  in  man.  Many 
of  them  are  of  the  same  general  size  and  shape  as  the  tubercle  bacillus, 
and,  strangely  enough,  produce  in  animals  small  diseased  areas  which 
not  only  macroscopically  but  also  microscopically  resemble  miliary 
tubercles  due  to  the  tubercle  bacillus.  They  are,  however,  entirely 
different  in  their  culture  characteristics,  producing  in  twenty-four  to 
forty-eight  hours,  on  ordinary  culture  media,  moist,  round  colonies  of 
an  eighth  to  a  quarter  of  an  inch  in  diameter,  and  of  a  more  or  less 
intense  pink  color.  In  animals  they  produce  only  localized  lesions, 
causing  death  only  when  injected  in  large  numbers.  The  injected 
animals  are  unaffected  by  tuberculin  injections.  The  chief  interest 
which  these  bacilli  have  for  us  is  the  possibility  of  confusing  them  with 
the  tubercle  bacilli.  This  danger  is  always  present  in  milk,  for  the  grass 
bacilli  find  so  many  means  of  gaining  entrance  to  it.  In  the  examination 
of  dust,  healthy  throat  and  nose  secretions,  etc.,  the  simple  micro- 
scopic examination  might  lead  to  error. 

They  can  be  separated  from  tubercle  bacilli  by  inoculating  animals 
in  which  no  progressive  lesions  will  develop.  If  there  is  any  doubt 
about  the  nature  of  the  infection,  inject  \  c.c.  of  tuberculin,  when  if 
infected  with  tuberculosis  they  will  die,  but  if  by  grass  bacilli  they  will 
show  no  reaction.  Cultures  from  the  lesions  will  also  show,  on  ordinary 
media,  pink  colonies  if  grass  bacilli  are  present,  and  no  growth  if  only 
tubercle  bacilli. 


CHAPTER   XXIII. 

THE   INFLUENZA  AND  PSEUDOINFLUENZA  BACILLI— THE   KOCH- 
WEEKS  BACILLUS. 

The  Influenza  Bacillus.— Influenza  as  a  distinct  entity  can  be 
traced  back  to  the  fifteenth  century  and  probably  existed  at  a  much 
earlier  date. 

At  times  but  few  endemic  cases  occur  and  then  a  great  epidemic 
spreads  over  the  civilized  world.  The  last  great  epidemic  reached 
Russia  from  the  East  in  the  fall  of  1889  and  gradually  spread  over 
Europe  and  to  America,  reaching  the  latter  country  in  December  of 
that  year.  Since  then  we  have  had  more  or  less  of  it,  especially  during 
the  winter  months. 

FIG.  100 


Influenza  bacilli.    X  1100  diameters. 

The  rapidity  of  the  spread  of  epidemics  of  influenza  suggested  that 
persons  were  the  carriers  of  the  infection,  while  the  location  of  the 
disease  pointed  to  the  respiratory  tract  as  the  location  of  and  to  the 
expectoration  as  the  chief  source  of  infection  by  the  micro-organisms. 

After  numerous  unsuccessful  attempts,  during  the  epidemic  of  1889 
and  succeeding  vears,  to  discover  the  specific  cause  of  influenza,  Pfeiffer 
(1892)  succeeded  in  isolating  and  growing  upon  blood  agar  a  bacillus 
which  abounded  in  the  purulent  bronchial  secretion  of  patients  suffer- 
ing from  epidemic  influenza,  which  he  showed  was  the  probable  cause 
of  the  disease.  This  bacillus  was  not  found  upon  normal  respiratory 
mucous  membranes. 

21 


322  BACTERIA  PATHOGENIC  TO  MAN 

Morphology. — Very  small,  moderately  thick  bacilli  (0.2  to  0.3//  in 
thickness  to  0.5  to  1.5/*  in  length),  usually  occurring  singly  or  united 
in  pairs,  but  threads  or  chains  of  three,  four,  or  more  elements,  are 
occasionally  found.  No  capsule  has  been  demonstrated. 

Staining. — The  bacillus  stains  with  difficulty  with  the  ordinary  aniline 
colors — best  with  dilute  ZiehPs  solution  (water  9  parts  to  ZiehPs  solution 
1  part),  or  Loeffler's  methylene-blue  solution,  with  heat.  When  faintly 
stained  the  two  ends  of  the  bacilli  are  sometimes  more  deeply  stained 
than  the  middle  portion.  They  are  not  stained  by  Gram's  method. 

Biology. — An  aerobic,  non-motile  bacillus;  does  not  form  spores;  no 
growth  occurs  with  most  cultures  below  26°  C.,  or  above  34°  C.,  or  in 
the  entire  absence  of  oxygen. 

Cultivation. — This  bacillus  is  best  cultivated  at  37°  C.,  and  on  the 
surface  of  ordinary  nutrient  culture  media  containing  haemoglobin.  Plain 
or  glycerin  agar,  or  blood  serum  thinly  streaked  with  rabbit  or  human 
blood,  make  the  best  media  for  their  growth.  At  the  end  of  eighteen 
hours  in  the  incubator  very  small  circular  colonies  are  developed, 
which,  under  a  low  magnification  (100  diameters),  appear  as  shining, 
transparent,  homogeneous  masses,  and  even  under  a  No.  7  lens  scarcely 
show  at  all  the  individual  organisms.  Older  colonies  are  sometimes 
colored  yellowish-brown  in  the  centre.  A  characteristic  feature  of  the 
influenza  bacillus  is  that  the  colonies  tend  to  remain  separate  from  each 
other,  although  when  they  are  thickly  sown  in  a  film  of  moist  blood 
upon  nutrient  agar  they  may  become  confluent.  Transplantation  of 
the  original  culture  to  ordinary  agar  or  serum  cannot,  as  a  rule,  be 
successfully  performed,  owing  to  the  want  of  sufficient  haemoglobin; 
but  if  sterile  rabbit,  pigeon,  or  human  blood  be  added  to  these  media 
transplantation  may  be  indefinitely  performed,  provided  it  is  done 
every  three  or  four  days.  Cultures  may  remain  alive  up  to  seventeen 
days  in  the  ice-chest.  By  a  series  of  beautifully  carried  out  experiments 
Pfeiffer  showed  that  not  only  were  the  red  blood  cells  the  necessary 
part  of  the  blood  needed  for  the  growth  of  the  influenza  bacillus,  but 
that  it  was  the  haemoglobin  in  the  cells. 

In  bouillon  in  thin  layers,  to  which  blood  is  added,  a  good  develop- 
ment takes  place  if  there  is  free  access  of  oxygen. 

RESISTANCE  AND  LENGTH  OF  LIFE. — The  influenza  bacillus  is  very 
sensitive  to  desiccation;  a  pure  culture  diluted  with  water  and  dried 
is  destroyed  with  certainty  in  twenty-four  hours;  in  dried  sputum  the 
vitality,  according  to  the  completeness  of  drying,  is  retained  from 
twelve  to  forty-eight  hours.  It  does  not  grow,  but  soon  dies  in  water. 
The  thermal  death  point  is  60°  C.  with  five  minutes'  exposure  (Pfeiffer 
and  Beck).  In  blood-bouillon  cultures  at  20°  C.  they  retain  their 
vitality  for  from  a  few  days  to  two  or  three  weeks.  In  moist  sputum 
it  is  difficult  to  determine  the  duration  of  their  life,  since  the  other 
bacteria  overgrow  and  make  it  impossible  to  find  them.  It  is  probable 
that  they  can  remain  alive  for  at  least  two  weeks.  The  bacilli  are  very 
readily  killed  by  chemicals,  disinfectants,  and  succumb  to  boiling 
within  one  minute  and  to  60°  C.  within  ten  minutes. 


THE  INFLUENZA  BACILLUS  323 

Detection  of  the  Influenza  Bacillus  in  Sputum. — The  direct  microscopic 
examination  of  stained  smears  of  sputum  may  give  considerable  infor- 
mation as  to  the  probable  presence  of  influenza-like  bacilli.  The 
frequent  presence  of  other  influenza-like  bacilli  in  the  throat  secretions 
leads  to  so  much  doubt  that  it  is  advisable  from  the  start  to  make  use 
of  plate  cultures,  the  best  medium  being  nutrient  agar  freshly  smeared 
with  a  film  of  rabbit's  blood. 

Pathogenesis. — The  bacillus  of  influenza,  in  so  far  as  experiments 
show,  produces  a  disease  at  all  similar  to  influenza  only  in  monkeys 
and  to  a  less  extent  in  rabbits.  When  a  small  quantity  of  culture  on 
blood  agar,  twenty-four  hours  old,  suspended  in*  1  c.c.  of  bouillon,  was 
injected  intravenously  into  rabbits,  Pfeiffer  found  that  a  characteristic 
pathogenic  effect  was  produced.  The  first  symptoms  were  developed 
in  one  and  a  half  to  two  hours  after  the  injection.  The  animals  became 
extremely  feeble,  lying  flat  upon  the  floor,  with  their  limbs  extended, 
and  suffered  from  extreme  dyspnoea.  The  temperature  rose  to  41°  C. 
or  above.  At  the  end  of  five  or  six  hours  they  were  able  to  sit  up  on 
their  haunches  again,  and  in  twenty-four  hours  had  recovered.  Larger 
doses  caused  the  death  of  the  animals  inoculated.  These  results  are 
attributed  by  Pfeiffer  to  toxic  products  present  in  the  cultures,  and  in 
none  of  his  experiments  was  he  ever  able  to  obtain  effects  resembling 
septicremic  infection.  In  some  of  the  experiments  on  monkeys,  these 
animals,  when  cultures  were  rubbed  into  the  nasal  mucous  membrane, 
showed  a  febrile  condition,  lasting  for  a  few  days;  but  in  no  instance 
has  Pfeiffer  observed  a  multiplication  of  the  bacilli  introduced. 

The  cell  bodies  of  the  bacilli  seem  to  possess  considerable  pyogenic 
action. 

Immunity. — Possibly  an  immunity  for  a  short  period  against  the 
influenza  poison  may  be  established  after  an  attack.  At  least  in  three 
experiments  made  by  Pfeiffer  on  monkeys,  these  animals,  after  recover- 
ing from  an  inoculation  with  bacilli,  seemed  to  be  much  less  susceptible 
to  a  second  injection. 

Distribution  of  Influenza  Bacilli  in  the  Body. — In  patients  suffering 
from  influenza  the  bacilli  are  found  chiefly  in  the  nasal  and  bronchial 
secretions.  In  acute  uncomplicated  cases  they  may  be  observed  micro- 
scopically in  large  masses,  and  often  in  absolutely  pure  culture;  the 
green,  purulent  sputum  derived  from  the  bronchial  tubes  is  especially 
suitable  for  examination.  The  older  the  process  is,  the  fewer  free  bacilli 
will  be  found  and  the  more  frequently  will  they  be  seen  lying  within 
the  pus  cells,  instead  of  being  embedded  free  in  the  secretion  as  at  first. 
At  the  same  time  they  stain  less  readily  and  present  more  irregular 
and  swollen  forms.  Very  frequently  the  influenza  process  invades 
portions  of  the  lung  tissue.  In  severe  cases  a  form  of  pneumonia  is 
the  result,  which  is  lobular  and  purulent  in  character,  and  accompanied 
by  symptoms  which  may  be  somewhat  characteristic  for  influenza,  or, 
again,  almost  identical  with  bronchopneumonia  due  to  the  pneumo- 
coccus.  The  walls  of  the  bronchioles  and  alveolar  septa  become  drnx-ly 
infiltrated  with  leukocytes,  and  the  spaces  of  the  bronchial  tubes  and 


324  BACTERIA  PATHOGENIC  TO  MAN 

alveoli  become  filled.  The  influenza  bacilli  are  found  crowded  in  be- 
tween the  epithelial  and  pus  cells  and  also  penetrate  the  latter.  There 
may  be  partial  softening  of  the  tissues,  or  even  abscess  formation. 
Bacilli  are  found  in  fatal  cases  to  have  penetrated  from  the  bronchial 
tubes  not  only  into  the  peribronchitic  tissue,  but  even  to  the  surface 
of  the  pleura,  and  rarely  they  have  been  obtained  in  pure  cultures  in 
the  pleuritic  exudation.  The  pleurisy  which  follows  influenza,  however, 
is  usually  a  secondary  infection,  due  to  the  streptococcus  or  pneumo- 
coccus. 

Presence  of  Influenza  Bacilli  in  Chronic  Influenza  and  in  Tuberculosis.— 
Ordinarily  influenza  runs  an  acute  or  subacute  course,  and  not  infre- 
quently it  is  accompanied  by  mixed  infections  with  the  pneumococcus 
and  the  streptococcus.  Pfeiffer  was  the  first  to  draw  attention  to  certain 
chronic  conditions  depending  upon  the  influenza  bacillus.  Bacilli  may 
be  retained  in  the  lung  tissue  for  months  at  a  time,  remaining  latent 
a  while,  and  then  becoming  active  again,  with  a  resulting  exacerbation 
of  the  disease.  Consumptives  are  liable  to  carry  influenza  bacilli  for 
years  and  are  particularly  susceptible  to  attacks  of  influenza.  Williams, 
in  the  examination  of  sputa  in  cases  of  pulmonary  tuberculosis,  has 
found  abundant  influenza  bacilli  to  be  present  in  a  large  proportion  of 
the  samples  of  sputum  from  consumptives,  and  this  not  only  in  winter 
but  also  in  the  summer,  when  no  influenza  was  known  to  be  present 
in  New  York.  Taken  together  with  results  elsewhere,  this  indicates 
that  at  all  times  of  the  year  many  consumptives  carry  about  with 
them  influenza  bacilli,  and  that  very  likely  many  healthy  persons  as 
well  as  persons  suffering  from  bronchitis  also  harbor  a  few.  Given 
proper  climatic  conditions,  we  have  at  all  times  the  seed  to  start  an 
epidemic. 

The  influenza  bacillus  does  not  occur,  as  a  rule,  in  the  blood  and 
probably  does  not  develop  there.  It  is  found  at  times  in  otitis  media 
accompanying  influenza,  and  has  been  found  in  the  meninges  in  cases 
of  meningitis.  So  far  as  positive  results  have  shown,  influenza  would 
seem  to  be  almost  always  a  local  infection  confined  chiefly  to  the  air 
passages.  The  general,  cerebral,  gastric,  and  other  symptoms  produced 
are  due  to  the  absorption  of  the  toxic  products  of  the  specific  organism, 
these  poisons  being  particularly  active  in  their  effects  on  the  central 
nervous  system. 

The  discovery  of  this  bacillus  enables  us  to  explain  many  things, 
previously  unaccountable,  in  the  cause  of  epidemic  influenza.  We  now 
know,  from  the  inability  of  the  influenza  bacillus  to  exist  for  long 
periods  in  dust,  that  the  disease  is  not  transmissible  to  great  distances 
through  the  air.  We  also  know  that  the  infective  material  is  con- 
tained only  in  the  catarrhal  secretions.  Sporadic  cases,  or  the  sudden 
eruption  of  epidemics  in  any  localities  from  which  the  disease  has  been 
absent  for  a  long  time,  or  where  there  has  been  no  new  importation 
of  infection,  may  possibly  be  explained  by  assuming  that  the  bacilli, 
as  already  mentioned,  often  remain  latent  in  the  lungs  or  bronchial 
secretions  of  the  body  for  many  months,  and  perhaps  years,  and  then 


PSEUDOINFLUENZA  BACILLI  325 

become  active  again,  when  under  favorable  circumstances  they  may 
be  communicated  to  others.  The  bacteriological  diagnosis  of  influ- 
enza is  of  considerable  importance  for  the  identification  of  clinically 
doubtful  cases,  which,  from  their  symptoms,  may  be  mistaken  for 
grippe,  or  vice  versa.  Among  these  are  bronchitis,  pneumonia,  or 
tuberculosis.  Up  to  the  present  time  the  diagnosis  gives  us  little  help 
in  treatment. 

Tn  acute  uncomplicated  cases  the  probable  diagnosis  can  be  frequently 
made  by  microscopic  examinations  of  stained  preparations  of  the 
sputum,  there  being  present  enormous  numbers  of  small  bacilli,  at  first 
largely  free  and  later  largely  in  the  pus  cells.  In  chronic  cases  or  those 
of  mixed  infection  few  or  many  bacilli  may  be  found  and  the  culture 
method  may  be  necessary  to  give  even  a  probable  diagnosis.  The  bacil- 
lus of  influenza  is  not  readily  separated  by  its  morphological,  staining, 
and  cultural  peculiarities  from  other  bacteria  belonging  to  the  influenza 
group  and  at  present  it  is  almost  impossible  to  certainly  identify  it. 

The  Pseudoinfluenza  Bacillus. — This  bacillus  is  culturally  very  similar 
to  the  typical  influenza  bacillus,  but  may  be  distinguished  from  it  by 
its  larger  size  and  tendency  to  grow  out  into  long  threads.  It  is  not 
certain  but  that  it  is  a  form  of  the  influenza  bacillus. 

Whooping-cough  Bacillus. — In  this  disease  bacilli  are  regularly  found 
which  differ  but  slightly  from  the  influenza  bacilli.  They  produce, 
when  injected  in  animals,  agglutinins  which  are  specific  for  them,  but 
not  for  influenza  bacilli.  Both  bacilli  are  affected  by  the  same  group 
(Wollstein).  The  blood  o'f  those  suffering  from  whooping-cough 
agglutinates  the  whooping-cough  bacilli  but  not  the  influenza  bacilli. 
Further  investigation  is  required  to  establish  whether  they  are  the 
exciting  factor  of  the  disease.  The  Koch-Weeks  bacillus  is  also  very 
similar  to  the  influenza  bacillus. 

Relation  of  the  Clinical  Symptoms  to  the  Bacterial  Excitant. — There  is  no 
doubt  that  other  infections  are  also  included  under  the  clinical  forms  of 
influenza,  and  during  an  epidemic  of  bronchopneumonias,  irregular  types 
of  lobar  pneumonias,  and  cases  of  bronchitis  frequently  have  symptoms 
so  closely  alike  that  the  nature  of  the  bacteria  active  in  the  case  is  very 
frequently  different  from  that  supposed  by  the  clinician.  Thus  in  four 
consecutive  autopsies  examined  by  the  writer  the  influenza  bacillus  was 
found  almost  in  pure  culture  in  one  case  believed  to  be  due  to  the 
pneumococcus,  and  entirely  absent  in  two  of  the  three  believed  to  be 
due  to  it.  Except  for  these  examinations  the  clinician  would  be  of  the 
opinion  that  he  had  clearly  diagnosed  bacteriologically  the  cases, 
while  in  fact  he  had  been  wrong  in  three  of  the  four. 

The  striking  symptoms  in  acute  respiratory  diseases  are  frequently 
more  due  to  the  location  of  the  lesions  than  to  the  special  variety  of 
organisms  producing  them.  In  epidemics  of  influenza  there  are,  of 
course,  many  cases  which,  on  account  of  their  characteristic  symptoms, 
can  be  fairly  certainly  attributed  to  the  influenza  bacilli.  Even  under 
these  circumstances  error  may  be  made,  as,  for  instance,  two  cases  of 
apparently  typical  influenza  were  reported  in  a  household  and  both 


326  BACTERIA  PATHOGENIC  TO  MAN 

showed  a  total  absence  of  influenza  bacilli.     The  pneumococcus  was 
present  in  almost  pure  culture. 

Examination  of  Sputum  for  Influenza  Bacilli. — 1.  Sputum  coughed 
from  the  deeper  air  passages  and  not  from  throat  scraping  should  be 
used. 

2.  The  sputum  should  be  received  in  a  sterile  bottle,  which  should 
then  be  placed  immediately  in  cracked  ice. 

3.  Blood-agar  plates  should  be  made  by  dropping  a  drop  of  fresh  rab- 
bit's blood,  obtained  aseptically,  on  the  centre  of  a  hardened  agar  plate. 

4.  One  of  the  more  solid  masses  of  the  sputum  should  be  taken  from 
the  bottle  with  sterile  forceps  and  placed  on  a  plain  agar  plate.     A 
small  portion  of  this  mass  should  be  separated  with  a  sterile  platinum 
needle  and  drawn  through  the  blood  on  the  blood-agar  plate  from  the 
centre  out  in  different  directions.     The  larger  part  of  what  is  left  of 
this  small    portion  is  then  placed    in  a  similar  manner  over  a  second 
blood  agar,  and  from  this  to  a  third,  sterilizing  the  needle  between  the 
transfers.     The  plates  should  be  placed  in  the  thermostat  for  twenty- 
four  hours. 

5.  After  the  plates  are  planted  two  smears  should  be  made,   one 
stained  by  Gram  and  the  other  by  weak  carbol-fuchsin. 

6.  After  twenty-four  hours  the  plates  are  examined  under  low  power. 
The  influenza  colonies  use  up  the  hemoglobin,  and  in  parts  of  the 
blood-agar  plate  where  the  blood  is  of  right  thickness  such  colonies 
show  as  almost  clear  areas    surrounded  by  the  red    blood.     With  a 
higher  power  (No.  6  or  7  objective),  if  such  areas  seem  to  be  made  up 
of  fine  indefinite  granulations,  they  are  practically  sure  to  be  influenza 
colonies.      Most   influenza   colonies   are   more   highly   refractive   than 
other  light  colonies,  and  they  show  this  characteristic  best  when  they 
grow  on  the  edge  of  a  blood  mass.    Many  influenza  colonies  also  show 
heapings  in  the  centre.     Influenza  colonies  growing  away  from  the 
blood  cells  are  less  characteristic  in  appearance  and  less  easily  differ- 
entiated from  other  bacteria. 

7.  Fishings  from  the  influenza-like  colonies  should  be  planted  on 
blood-agar  tubes,  and  if,  after  twenty-four  hours  in  the  thermostat,  the 
resulting   growth  should  consist  of   influenza-like  organisms,  plantings 
should  be  made  on  plain  agar.     The  first  generation  on  plain  agar 
may  show  slight  growth  because  of  the  blood  carried  over  from  the 
original  tube,  but  the  second  generation  should  show  no  growth  if  the 
organism  is  the  influenza  bacillus. 

8.  The  agglutination  characteristics  of  the  cultures  should  be  tested 
in  the  serum  from  a  rabbit  injected  with  a  single  typical  culture,  and  in 
the  serum  from  one  injected  with  a  number  of  cultures.     The  aggluti- 
nation tests  should  be  carried  out  in  order  to  gain  information.     The 
cultures  tested  in  the  Research  Laboratory  have  shown  considerable 
variation. 

For  Testing  the  Agglutination  of  Influenza  Bacilli  in  the  Hanging  Drop. 
— Grow  the  cultures  on  slanted  agar  tubes  to  which,  after  cooling  to 
40°  C.,  i  c.c.  of  defibrinated  blood  has  been  added.  When  twenty  to 


KOCH-WEEKS  BACILLUS  327 

twenty-four  hours  old  make  a  suspension  of  the  bacilli  in  normal  salt 
solution,  controlling  the  number  of  bacilli  by  examining  a  hanging-drop 
preparation.  The  influenza  bacilli  seem  to  agglutinate  rather  slowly; 
so  it  usually  takes  four  to  five  hours  to  get  a  good  reaction. 

Serum  Therapeutics. — No  protective  serum  has  been  produced  which 
has  any  value  in  treatment. 

The  Koch-Weeks  Bacillus  of  Conjunctivitis. 

This  bacillus  was  first  observed  by  R.  Koch  in  1883  while  making 
certain  investigations  into  inflammation  of  the  eye  occurring  during 
an  epidemic  of  cholera  in  Alexandria.  It  was  later,  in  1887,  more 
specifically  described  by  Weeks  in  New  York.  Weeks  obtained  it  in 
pure  culture  in  1890. 

The  infective  disease  which  is  caused  by  this  bacillus  seems  to  be 
very  widely  distributed,  no  land  or  clime  probably  being  exempt  from 
it.  In  this  country  it  occurs  epidemically  and  with  increasing  fre- 
quency during  the  Spring  and  Fall  months.  Weeks  has  found  the 
bacillus  in  over  1000  cases  in  New  York  City. 

Morphology. — The  bacilli  from  the  purulent  secretions  are  small  and 
slender,  being  not  unlike  the  influenza  bacilli  but  somewhat  longer. 
They  vary  in  length  from  0.5  to  I/JL  or  even  2/t  occasionally;  the 
longer  forms  are  apparently  unions  of  thread-like  filaments.  The 
shorter  bacilli  not  infrequently  have  the  appearance  of  diplococci. 
Sometimes  they  exhibit  slight  polar  staining.  Their  width  is  very 
constant.  The  ends  are  rounded.  They  are  rapidly  decolorized  by 
Gram. 

Staining. — They  are  best  stained  by  very  dilute  solutions  of  carbol- 
fuchsin  or  LoefHer's  methylene  blue,  but  do  not  stain  readily. 

In  smear  preparations  the  Koch- Weeks  bacilli  are,  as  a  rule,  seen 
alone  or  associated  with  isolated  cocci  and  bacilli,  especially  xerosis 
bacilli.  They  are  not  infrequently  observed  within  the  cells,  and  are 
very  rarely  associated  with  gonococci  and  pneumococci,  such  mixed 
infections  being  extremely  uncommon. 

Biological  Characters. — The  Koch- Weeks  bacillus  grows  only  at  incu- 
bator temperature.  Of  the  ordinary  culture  media  none  but  moist 
and  slightly  alkaline  peptone  agar  can  be  employed.  Special  media 
are  required  generally  for  its  cultivation.  The  best  results  have  been 
obtained  with  serum  agar  or  a  mixture  of  glycerin  agar  and  ascitic 
fluid,  2  to  1.  Pure  cultures  are  rarely  obtained  at  first;  they  are  usually 
associated  with  colonies  of  xerosis  bacilli  or  staphylococci.  After 
twenty-four  to  forty-eight  hours  the  colonies  are  noticeable  as  moist, 
transparent,  shining  drops  or  points.  Microscopically  examined  under 
low  magnifying  power  they  appear  like  small  gas  bubbles;  by  closer 
examination  they  are  seen  to  be  round,  lying  loosely  on  the  surface, 
and  are  readily  removed.  Under  higher  magnification  a  number  of 
fine  points  are  observable.  The  colonies,  which  resemble  those  of 
influenza,  have  a  tendency  to  confluesce,  but  are  not  so  sharply  defined 


328  BACTERIA  PATHOGENIC  TO  MAN 

as  the  latter  and  become  more  quickly  indistinguishable.  Isolated 
colonies,  especially  those  in  the  neighborhood  of  xerosis  bacilli  or 
staphylococci,  grow  larger  and  their  contour  is  slightly  wavy;  they  are 
more  opaque  and  granular  than  influenza  colonies.  In  serum  or 
blood  bouillon  a  slight  cloudiness  is  produced  which  finally  settles 
down. 

Resistance. — In  culture  media  the  bacilli  die  rapidly,  seldom  living 
more  than  five  days.  Development  ceases  at  20°.  They  resist  a 
temperature  of  50°  for  ten  minutes,  but  cannot  withstand  60°  for  more 
than  one  or  two  minutes.  Kept  for  one  and  a  half  hours  at  — 7°  they 
still  remain  alive.  Exposure  to  the  sun's  rays  for  one-half  hour  does 
not  kill  them,  but  at  the  end  of  two  and  one-half  hours  they  die.  They 
cannot  resist  dying  for  any  length  of  time. 

Transmission. — This  occurs  only  by  contact  either  by  direct  or  indirect 
conveyance  of  the  moist  infective  material.  Infection  is  not  communi- 
cated through  the  air  by  means  of  dust,  as  the  bacilli  soon  die  when 
dried.  It  may,  however,  be  conveyed  by  flies,  etc. 

Pathogenesis. — The  Koch- Weeks  bacillus  is  not  pathogenic  for  ani- 
mals. Man,  on  the  contrary,  is  extremely  susceptible  to  infection  from 
this  bacillus,  which  produces  one  of  the  most  contagious  diseases 
known. 

Immunity  is  not  produced  to  any  extent  by  one  attack,  but  there 
does  seem  to  be  an  individual  susceptibility  to  the  disease. 

Differential  Diagnosis. — The  only  micro-organisms  from  which  the 
Koch-Weeks  bacillus  would  seem  to  require  differentiation  are  the 
influenza  bacillus  of  Pfeiffer,  the  so-called  influenza  bacillus  of  con- 
junctivitis of  Miiller  and  the  pseudoinfluenza  bacillus  of  Zur  Nedden. 
These  latter  bacilli,  however,  grow  well  only  on  hsemoglobin  media, 
which  the  Koch- Weeks  bacillus  does  not  require.  The  colonies  on 
serum  agar  are  smaller  than  those  of  the  influenza  bacilli  and  the 
edges  are  more  granular.  While  the  influenza  bacillus  is  slightly 
pathogenic  for  certain  animals,  the  Koch-Weeks  bacillus  has  so  far 
given  negative  results  with  all  animals.  Clinically  also  the  disease  is 
distinctly  different. 


CHAPTER  XXIV. 

THE  PRODUCERS  OF  ABSCESSES,  CELLULITIS,  SEPTICAEMIA.  ETC. 
THE  STAPHYLOCOCCI. 

STAPHYLOCOCCI  were  first  obtained  from  pus  by  Pasteur  in  1880. 
In  1881  Ogston  showed  that  they  frequently  occurred  in  abscesses,  and 
in  1884  Rosenbach  fully  demonstrated  their  etiological  importance  in 
•circumscribed  abscesses,  osteomyelitis,  etc.  Of  all  the  staphylococcus 
varieties  the  staphylococcus  pyogenes  aureus  is  by  far  the  most  important 
and  will,  therefore,  be  first  described. 

The  Staphylococcus  Pyogenes  Aureus. 

The  staphylococcus  pyogenes  aureus  is  one  of  the  commonest  patho- 
genic bacteria,  being  usually  present  in  the  skin  or  mucous  membranes, 
and  is  the  organism  most  frequently  concerned  in  the  production  of 
acute,  circumscribed,  suppurative  inflammations. 

Morphology. — Small,  spherical  cells,  having  a  diameter  of  0.7/*  to  0.9//, 
occurring  solitary,  in  pairs  as  diplococci,  in  short  rows  of  three  or 
four  elements,  or  in  groups  of  four,  but  most  commonly  in  irregular 
masses,  simulating  clusters  of  grapes;  hence  the  name  staphylococcus. 
(See  Fig.  101.) 

Staining. — It  stains  quickly  in  aqueous  solutions  of  the  basic  aniline 
colors  and  with  many  other  dyes.  When  previously  stained  with 
gentian  violet  it  is  not  decolorized  by  Gram's  method.  When  slightly 
stained  each  sphere  frequently  is  seen  to  be  already  dividing  into  two 
semispherical  bodies. 

Biology. — The  staphylococcus  pyogenes  aureus  is  an  aerobic,  /ac?//- 
tative  anaerobic  micrococcus,  growing  at  a  temperature  from  8°  to 
43°  C.,  but  best  at  25°  to  35°  C.  The  staphylococci  grow  readily  on  all 
the  common  laboratory  media,  such  as  milk,  bouillon,  nutrient  gelatin, 
or  agar.  A  slightly  alkaline  reaction  to  litmus  is  best  for  the  growth 
of  the  staphylococci,  but  they  also  grow  in  slightly  acid  media. 

Cultivation.  GROWTH  ix  NUTRIENT  BOUILLON. — The  growth  of  the 
staphylococcus  is  rapid,  reaching  about  50,000,000  per  c.c.  at  the  end 
of  twenty-four  hours  at  30°  C.  The  bouillon  is  cloudy  and  frequently 
has  a  thin  pellicle.  Later  a  shiny  sediment  forms.  The  odor  is  dis- 
agreeable. In  peptone-water  growth  occurs  with  indol  production. 

GROWTH  ON  GELATIN. — Grown  on  gelatin  plates  it  develops,  at  room- 
temperature,  within  forty-eight  hours,  punctiform  colonies,  which,  when 
examined  under  a  low-power  lens,  appear  as  circular  disks  of  a  pale- 


330 


BACTERIA  PATHOGENIC  TO  MAN 


FIG.  101 


Staphylococcus.    X  1100  diameters. 


brown  color,  somewhat  darker  in  the  centre,  and  surrounded  by  a 
smooth  border.  The  colonies  grow  rapidly.  The  appearance  of  the 
growth  is  most  characteristic.  Immediately  surrounding  the  colonies, 
which  are  of  a  pale-yellow  color,  there  is  a  deepening  of  the  surface  of 
the  gelatin,  due  to  its  liquefaction.  By  suitable  light  a  number  of  these 
shallow  depressions  with  sharply  defined  outlines  may  be  seen  on  the 
gelatin  plate,  having  a  diameter  of  from  5  to  10  mm.,  in  the  centres  of 

which  lie  the  yellow  colonies.  Later,  the 
liquefaction  becomes  general,  the  colonies 
running  together.  In  stab  cultures  in 
gelatin  a  white  confluent  growth  at  first 
appears  along  the  line  of  puncture,  followed 
by  liquefaction  of  the  medium,  which 
rapidly  extends  to  the  sides  of  the  test- 
tube.  At  the  end  of  two  days  the  yellow 
pigmentation  begins  to  form,  and  this  in- 
creases in  intensity  for  eight  days.  Finally, 
the  gelatin  is  completely  liquefied,  and  the 
staphylococci  form  a  golden-yellow  or 
orange-colored  deposit  at  the  bottom  of  the 
tube.  Under  unfavorable  conditions  the 
Staphylococcus  aureus  gradually  loses  its  ability  to  make  pigment  and 
to  liquefy  gelatin. 

GROWTH  ON  AGAR. — In  streak  and  stab  cultures  on  agar  a  whitish 
growth  is  at  first  produced,  and  this  at  the  end  of  a  few  days  becomes  a 
faint  to  a  rich  golden-yellow  on  the  surface.  The  yellow  pigmentation 
is  produced  only  in  the  presence  of  oxygen ;  colonies  found  at  the  bottom 
of  a  stab  culture  or  under  a  layer  of  oil  remain  white. 

MILK. — Milk  inoculated  with  this  micrococcus  is  coagulated  at  the 
end  of  from  one  to  eight  days. 

GROWTH  ON  POTATO. — Upon  this  substance  the  staphylococci  grow 
readily  and  produce  abundant  pigment. 

GROWTH  ON  LOEFFLER'S  SOLIDIFIED  BLOOD  SERUM. — Growth  vigor- 
ous, with  fairly  good  pigment  production.  Some  varieties  slowly  liquefy 
the  serum. 

GROWTH  ON  BLOOD  AGAR. — If  nutrient  agar  to  which  a  little  animal 
blood  has  been  added  is  streaked  with  staphylococci  there  appears,  at 
the  end  of  twenty-four  hours  at  35°  C.,  about  the  growth  a  clear  zone, 
owing  to  the  hsemolytic  effect  of  the  Staphylococcus  products. 

In  certain  culture  media,  as  the  result  of  the  growth  of  the  Staphylo- 
coccus aureus,  there  is  a  production  of  acid  in  considerable  quantities, 
these  consisting  chiefly  of  lactic,  butyric,  and  valerianic  acids.  These 
acids  have  been  supposed  to  play  a  part  in  the  production  of  pus,  in 
which,  according  to  some  observers,  they  are  often  present. 

RESISTANCE. — The  Staphylococcus  is  distinguished  from  most  other 
pathogenic  bacteria  by  its  comparatively  greater  power  of  resistance 
to  outside  influences,  desiccation,  etc.,  as  well  as  to  chemical  disinfectants. 
Cultures  of  the  Staphylococcus  pyogenes  in  gelatin  or  agar  retain  their 


PRODUCERS  OF  ABSCESSES.  (  I.LLULIT1S  AXD  SEPT  K.  I-  MI.\     331 

vitality  for  a  year  or  more.  Suspended  in  water  its  thermal  death  point 
varies  with  different  cultures  and  averages  about  two  hours  at  50°  C.,  one- 
half  to  one  hour  at  60°  C.,  ten  minutes  at  70°  C.,  and  five  minutes  at 
80°  C.  Upon  silk  threads  and  in  media  rich  in  organic  matter  its 
resistance  is  greater,  but  subjected  to  80°  C.  for  thirty  minutes  or  boiling 
for  two  minutes  it  is  almost  surely  killed.  Cold  has  but  little  effect. 
Thirty  per  cent,  of  the  organisms  remained  alive  after  being  subjected 
by  us  to  freezing  in  liquid  air  for  thirty  minutes. 

They  are  quite  resistant  to  direct  sunlight  and  to  drying.  Dried  pus 
contains  living  staphylococci  for  weeks  and  even  months,  and  they  can 
be  found  alive  in  the  fine  dust  of  the  air  in  living  and  in  operating  rooms. 

EFFECT  OF  CHEMICALS. — In  water  they  remain  alive  for  several  weeks. 
To  most  disinfectants  the  staphylococci  are  rather  resistant.  The 
presence  with  staphylococci  of  organic  substances,  especially  albumin, 
increases  their  resistance.  In  watery  solution  dissolved  mercuric 
chloride,  1 : 1000,  destroys  the  organisms  in  five  to  fifteen  minutes, 
but  when  in  pus  not  for  several  hours. 

Hydrogen  peroxide  in  1  per  cent,  solution  kills  in  about  one-half  hour. 
Methyl  alcohol  in  50  per  cent,  solution  kills  staphylococci  on  silk  threads 
in  ten  minutes.  The  same  effect  is  obtained  by  carbolic  acid  in  3  per 
cent,  solution  or  lysol  in  1  per  cent,  solution.  Formaldehyde  in  watery 
solution  acts  only  in  concentrations  of  5  per  cent,  or  over. 

P  thoren'sis. — The  pathogenic  effect  of  the  staphylococcus  pyogenes 
aureus  on  test  animals  varies  considerably,  according  to  the  mode  of 
application  and  the  virulence  of  the  special  culture  employed.  In  the 
experiments  so  far  made  this  micrococcus,  as  found  in  suppurative 
processes  in  the  human  subject,  has  not  proved  to  be  as  infectious  for 
animals  as  it  is  for  man.  In  man  a  simple  rubbing  of  the  surface  of  the 
unbroken  skin  with  pus  from  an  acute  abscess  is,  as  a  rule,  sufficient 
to  produce  purulent  inflammation,  and  the  introduction  of  a  few  germs 
from  a  septic  case  into  a  wound  may  lead  to  a  fatal  pysemia.  These 
conditions  can  only  be  reproduced  in  lower  animals  with  difficulty,  and 
by  the  inoculation  of  large  quantities  of  the  culture.  Subcutaneous 
injections,  or  the  inoculation  of  open  wounds  in  mice,  guinea-pigs,  and 
rabbits,  are  commonly  without  result;  occasionally  abscess  formation 
may  follow  at  the  point  of  inoculation,  which  usually  ends  in  recovery. 
The  pus-producing  property  of  the  organism  is  exhibited  in  proportion 
to  the  virulence  of  the  culture  employed.  Slightly  virulent  cultures, 
which  constitute  the  majority  of  those  obtained  from  pus  taken  from 
the  human  subject,  when  injected  subcutaneously  in  large  quantities 
(several  c.c.  of  a  fresh  bouillon  culture)  in  rabbits  or  guinea-pigs,  give 
rise  to  local  pathological  lesions- -acute  abscesses.  When  virulent 
cultures  are  used — which  are  rarely  obtainable — 0.5  c.c.  of  a  fresh 
bouillon  culture  is  sufficient  to  produce  similar  results.  The  abscesses 
generally  heal  without  treatment;  sometimes  the  animals  die  from 
marasmus  in  consequence  of  the  suppurative  process.  In  intraperitoneal 
inoculations  the  degree  of  virulence  of  the  culture  employed  is  still 
more  conspicuous  in  the  effects  produced.  The  animals  usually  die  in 


332  BACTERIA  PATHOGENIC  TO  MAN 

from  two  to  nine  days.  The  most  characteristic  pathological  lesions  are 
found  in  the  kidneys,  which  contain  numerous  small  collections  of  pus, 
and  under  the  microscope  present  the  appearances  resulting  fromembolic 
nephritis.  Punctiform,  whitish-yellow  masses  of  the  size  of  a  pea  are 
found  permeating  the  pyramids.  Many  of  the  capillaries  and  some  of 
the  smaller  arteries  of  the  cortex  are  plugged  up  with  thrombi,  consisting 
of  micrococci.  Metastatic  abscesses  may  also  be  observed  in  the  joints 
and  muscles.  The  micrococci  may  be  recovered  in  pure  cultures  from 
the  blood  and  the  various  organs;  but  they  are  not  numerous  in  the 
blood  and  are  often  difficult  to  demonstrate  microscopically.  Intra- 
venous inoculations  of  animals  are  followed  by  similar  pathological 
changes.  Orth  and  Wyssokowitsch  first  pointed  out  that  injection  of 
staphylococci  into  the  circulation  of  rabbits  whose  cardiac  valves  have 
previously  been  injured  produced  ulcerative  endocarditis.  Subsequently, 
Weichselbaum,  Prudden,and  Fraenkel  and  Sanger  obtained  confirmatory 
results,  thus  establishing  the  fact  that  when  the  valves  are  first  injured, 
mechanically  or  chemically,  the  injection  into  a  vein  of  a  pure  culture  of 
staphylococcus  aureus  gives  rise  to  a  genuine  ulcerative  endocarditis.  It 
has  been  further  shown  by  Ribbert  that  the  same  result  may  be  obtained 
without  previous  injury  to  the.valves  by  injecting  into  a  vein  the  staphylo- 
coccus from  a  potato  culture  suspended  in  water.  In  his  experiments 
not  only  the  micrococci  from  the  surface  but  the  superficial  layer  of  the 
potato  were  scraped  off  with  a  sterilized  knife  and  mixed  with  distilled 
water,  and  the  successful  result  is  ascribed  to  the  fact  that  the  little 
agglomerations  of  micrococci  and  infected  fragments  of  potato  attach 
themselves  to  the  margins  of  the  valves  more  readily  than  isolated  cocci 
would  do.  Not  infrequently,  also,  in  intravenous  inoculations  of  young 
animals  there  occurs  a  localization  of  the  injected  material  in  the  marrow 
of  the  small  bones.  This  may  take  place  in  full-grown  animals  when 
the  bones  have  been  injured  or  fractured.  The  experimental  osteo- 
myelitis thus  produced  has  been  demonstrated  to  be  anatomically  analo- 
gous to  this  disease  in  man.  With  regard  to  the  lesions  found  in  the 
kidneys  after  intraperitoneal  or  intravenous  inoculation  of  cultures  of 
the  staphylococcus,  it  has  been  found  that  when  injected  in  considerable 
quantities  the  organism  may  be  obtained  in  cultures  from  the  urine, 
but  not  sooner  than  six  or  eight  hours  after  the  injection,  and  not  until 
the  formation  of  purulent  foci  in  the  kidneys  has  already  occurred. 

Toxic  SUBSTANCES. — Filtrates  of  cultures  contain  very  toxic  sub- 
stances. Injected  into  the  peritoneal  cavity  they  excite  peritonitis. 
Under  the  skin  they  produce  infiltration  or  abscess.  In  the  blood 
they  injure  both  the  red  and  white  corpuscles. 

Cultures  of  the  staphylococcus,  when  sterilized  by  boiling  and  in- 
jected subcutaneously,  produce  marked  positive  chemotaxis  and  often 
local  abscesses.  Leber  found  also  that  sterilized  cultures  introduced 
into  the  anterior  chamber  of  the  rabbit's  eye  would  bring  about  a  fibro- 
purulent  inflammation,  the  cornea  becoming  infiltrated,  and  perforation 
alongside  of  the  sclerotic  ring  finally  taking  place.  This  was  followed 
by  the  formation  of  pus  in  the  anterior  chamber  and  recovery.  These 


PRODUCERS  OF  ABSCESSES,  CELLULITIS  AXD  SEPTICAEMIA     333 

local  changes  follow  the  inoculation  of  small  (juantities  only  of  the 
dead  cultures;  but  when  large  amounts  are  injected  into  a  vein  or 
into  the  abdominal  cavity,  toxic  effects  are  produced.  Dogs  and 
guinea-pigs  thus  treated  usually  die,  showing  symptoms  of  poisoning. 
The  hn'inolytic  effect  of  certain  products  of  virulent  staphylococci  have 
recently  been  studied.  In  cultures  they  can  be  detected  about  the  third 
or  fourth  day  and  reach  their  maximum  on  the  ninth  to  fourteenth 
day.  Virulent  staphylococci  are  more  apt  to  produce  this  substance 
than  the  non-virulent,  but  there  is  no  definite  rule. 

A  poison  which  injures  leukocytes  is  also  produced.  This  is  destroyed 
at  about  57°  C. 

Other  poisons  of  less  definite  properties  are  also  found  in  the  filtrate. 

Immunization. — Immunity  against  staphylococcus  infection  may  be 
produced  in  different  animal  species  by  the  injection  of  increasing 
doses  of  the  pure  culture,  either  living  or  previously  sterilized  by  heating. 
Wright  claims  to  have  injected  dead  cultures  with  good  results  in  persons 
suffering  from  staphylococcus  skin  infections. 

The  blood  serum  of  animals  which  have  been  immunized  by  means 
of  living  or  dead  cultures  possesses  slight  immunizing  and  curative 
effects  in  other  animals,  but  no  practical  use  of  the  serum  has  been 
attempted  in  man. 

Occurrence  in  Man. — Practically  all  micro-organisms  have  been  shown 
by  experiment  to  produce,  under  certain  conditions,  the  formation  of 
pus  by  their  products  when  inoculated  into  the  animal  body ;  but,  while 
this  has  been  demonstrated,  the  researches  of  bacteriologists  show 
that  only  a  few  species  are  usually  concerned  in  the  production  of  acute 
abscesses  in  man.  Of  these  the  two  most  important,  by  reason  of 
their  frequent  occurrence  and  pathogenic  power,  are  staphylococcus 
pyogenes  and  streptococcus  pyogenes.  These  two  organisms  are  often 
found  in  the  same  abscess;  thus,  Passet,  in  33  cases  of  acute  abscess, 
found  staphylococcus  aureus  and  albus  associated  in  11,  albus  alone 
in  4,  albus  and  citreus  in  2,  streptococcus  pyogenes  alone  in  8,  albus 
and  streptococcus  in  1,  and  albus,  citreus,  and  streptococcus  in  1.  The 
staphylococcus  is  liable  to  enter  as  a  mixed  infection  into  most  infec- 
tions due  to  other  bacteria,  and  is  almost  always  met  with  in  all  inflam- 
mations of  the  skin  and  mucous  membranes  or  in  cavities  connected 
with  them.  Care  must  always  be  taken  that  the  simple  finding  of 
staphylococci  does  not  cause  one  to  overlook  other  organisms,  which 
perhaps  were  the  original  exciters  of  the  diseased  process. 

As  the  result  of  extended  researches  the  golden  staphylococcus  has 
been  demonstrated  not  only  in  furuncles  and  carbuncles,  but  also  in 
various  pustular  affections  of  the  skin  and  mucous  membranes — 
impetigo,  sycosis,  phlyctenular  conjunctivitis;  in  purulent  conjunctivitis 
and  inflammation  of  the  lacrymal  sac;  in  acute  abscesses  formed  in  the 
lymphatic  glands,  the  parotid  gland,  the  tonsils,  the  mamma?,  etc.;  in 
metastatic  abscesses  and  purulent  collections  in  the  joints;  in  empyema, 
infectious  osteomyelitis,  ulcerative  endocarditis,  pyelonephritis,  abscess 
of  the  liver,  phlebitis,  etc.  It  is  one  of  the  chief  etiological  factors 


THF 
IIKMX/FRP 


-• 

S'.TY    1 


334  BACTERIA  PATHOGENIC  TO  MAN 

in  the  production  of  pysemia  in  the  various  pathological  forms  of 
that  condition  of  disease.  It  is  remarkable  how  many  staphylococci 
may  he  p  esent  in  the  blood  without  a  fatal  result,  if  the  original  source 
of  infection  is  removed.  We  met  with  one  case  in  which  over  800 
staphylococci  were  present  in  1  c.c.  of  blood.  A  week  later  only  five 
were  found.  The  patient  finally  died  from  pneumonia. 

Not  all  persons  are  equally  susceptible  to  infection  by  the  staphy- 
lococcus;  those  who  are  in  a  cachectic  condition  or  suffering  from  con- 
stitutional diseases,  like  diabetes,  are  especially  predisposed  to  infec- 
tion. In  healthy  individuals  certain  parts  of  the  body,  as  the  back  of 
the  neck  and  the  buttocks,  are  more  liable  to  be  attacked  than  others, 
with  the  production  of  furuncles,  carbuncles,  etc.  In  persons  in  whom 
sores  are  readily  caused,  in  consequence  of  disturbances  of  nutrition, 
as  in  exhausting  diseases,  the  micrococci  settle  at  the  points  of  least 
resistance.  Such  conditions  are  present  in  the  bones  of  debilitated 
young  children,  in  fractures,  and  injuries  in  general. 

The  pyogenic  properties  of  the  staphylococcus  have  been  demon- 
strated upon  man  by  numerous  experiments.  Garre  inoculated  a  small 
wound  at  the  edge  of  one  of  his  finger-nails  with  a  minute  quantity  of 
a  pure  culture,  and  purulent  inflammation,  extending  around  the  mar- 
gin of  the  nail,  resulted  from  the  inoculation.  Staphylococcus  aureus 
was  recovered  in  cultures  from  the  pus  thus  formed.  The  same  observer 
applied  a  considerable  quantity  of  a  pure  culture  obtained  from  this 
pus — third  generation — to  the  unbroken  skin  of  his  forearm,  rubbing 
it  well  into  the  skin.  At  the  end  of  four  days  a  large  carbuncle,  sur- 
rounded by  isolated  furuncles,  developed  at  the  point  where  the  culture 
had  been  applied.  This  ran  the  usual  course,  and  it  was  several  weeks 
in  healing.  No  less  than  seventeen  scars  remained  to  testify  to  the 
success  of  the  experiment. 

Staphylococcus  Pyogenes  Albus. 

It  is  morphologically  identical  with  the  staphylococcus  pyogenes 
aureus,  and  is  probably  the  same  organism  which  has  lost  the  property 
of  producing  pigment.  On  the  average  it  is  somewhat  less  pathogenic 
and  seldom  produces  pyaemia  or  grave  infections.  The  surface  cul- 
tures upon  nutrient  agar  and  potato  have  a  milk-white  color.  Its 
biological  characters  are  not  to  be  distinguished  from  the  staphylo- 
coccus aureus. 

The  majority  of  bacteriologists  agree  with  Rosenbach,  that  the 
aureus  is  found  at  least  twice  as  frequently  in  human  pathological 
processes  as  the  albus. 

Staphylococcus  Epidermidis  Aibus  (Welch). 

Probably  identical  with  the  staphylococcus  pyogenes  albus.  With 
reference  to  this  micrococcus,  Welch  says:  "So  far  as  our  observations 
extend — and  already  they  amount  to  a  large  number — this  coccus  may 


PRODUCERS  OF  ABSCESSES,  CELLULITIS  AXD  SEPTICAEMIA     335 

be  regarded  as  nearly,  if  not  quite,  a  constant  inhabitant  of  the  epider- 
mis. It  is  now  clear  \\liy  I  have  proposed  to  call  it  the  staphylococcus 
epidermidis  albus.  It  possesses  such  feeble  pyogenic  capacity,  as  is 
shown  by  its  behavior  in  wounds,  as  well  as  by  experiments  on  rabbits, 
that  the  designation  staphylococcus  pyogenes  albus  does  not  seem 
appropriate.  Still,  I  am  not  inclined  to  insist  too  much  upon  this 
point,  as  very  probably  this  coccus — which  has  hitherto  been  unques- 
tionably identified  by  others  with  the  ordinary  staphylococcus  pyogenes 
albus  of  Rosenbach — is  an  attenuated  or  modified  form  of  the  latter 
organism,  although,  as  already  mentioned,  it  presents  some  points  of 
difference  from  the  classical  description  of  the  white  pyogenic  coccus." 
According  to  Welch,  this  coccus  differs  from  the  staphylococcus 
albus  in  the  fact  that  it  liquefies  gelatin  more  slowly,  does  not  so  quickly 
cause  coagulation  in  milk,  and  is  far  less  virulent  when  injected  into 
the  circulation  of  rabbits.  It  has  been  shown  by  the  experiments  of 
Bossowski  and  of  Welch  that  this  micro-organism  is  very  frequently 
present  in  aseptic  wounds,  and  that  usually  it  does  not  materially  inter- 
fere with  the  healing  of  wounds,  although  sometimes  it  appears  to 
cause  suppuration  along  the  drainage  tube,  and  it  is  the  common  cause 
of  "stitch  abscess." 

Staphylococcus  Pyogenes  Citreus  and  other  Staphylococci. 

Isolated  by  Passet  (1885)  from  the  pus  of  acute  abscesses,  in  which 
it  is  occasionally  found  in  association  with  other  pyogenic  cocci.  It 
is  distinguished  from  the  other  species  only  by  the  formation  of  a  lemon- 
yellow  pigment. 

Many  other  varieties  of  staphylococci  have  been  occasionally  met 
with  which  differ  in  some  respects  from  the  typical  varieties.  This  differ- 
ence may  be  in  the  fact  that  they  liquefy  gelatin  more  slowly  or  not  at  all, 
or  in  pigment  formation,  or  in  agglutination,  or  in  still  other  respects. 
None  of  these  varieties  are  of  great  importance. 

The  Micrococcus  Tetragenus. 

This  organism  was  discovered  by  Gaffky  (1881).  It  is  not  infre- 
quently present  in  the  saliva  of  healthy  individuals  and  in  the  sputum 
of  consumptive  patients.  In  sputum  it  is  sometimes  an  evidence  of 
mouth  contamination  rather  than  lung  infection.  It  has  repeatedly 
been  observed  in  the  walls  of  cavities  in  pulmonary  tuberculosis  asso- 
ciated with  other  pathogenic  bacteria,  which,  though  playing  no  part 
in  the  etiology  of  the  original  disease,  contribute,  doubtless,  to  the 
progressive  destruction  of  the  lung.  Its  pyogenic  character  is  shown 
by  its  occasional  occurrence  in  the  pus  of  acute  abscesses.  Its  presence 
has  also  been  noted  in  the  pus  of  empyema  following  pneumonia. 

Morphology. — Micrococci  having  a  diameter  of  about  I/*,  which 
divide  in  two  directions,  forming  tetrads,  and  bound  together  by  a  tran^- 
parent,  gelatinous  substance,  enclosing  the  cell  like  a  capsule.  In 
cultures  the  cocci  are  seen  in  various  stages  of  division  as  large,  round, 


336  BACTERIA  PATHOGENIC  TO  MAN 

undivided  cells,  in  pairs  of  oval  elements,  and  in  groups  of  three  and 
four  (Fig.  102).  When  the  division  is  complete  they  remind  one  of 
sarcina?  in  appearance,  except  that  they  do  not  divide  in  three  direc- 
tions and  are  not  built  up  like  diminutive  cotton  bales. 

Staining. — This  micrococcus  stains  readily  with  the  ordinary  aniline 
dyes;  the  transparent  gelatinous  envelope  is  only  feebly  stained.  It  is 
not  decolorized  by  Gram's  method. 

Biology. — The  growth  of  this  micrococcus  is  slow  under  all  condi- 
tions. It  grows  both  in  the  presence  and  absence  of  oxygen;  it  grows 
best  from  35°  to  38°  C.,  but  may  be  cultivated  also  at  the  ordinary 
room-temperature — about  20°  C. 

Growth  on  Gelatin. — On  gelatin  plates  small,  white  colonies  are 
developed  in  from  twenty-four  to  forty-eight  hours,  which,  when  exam- 
ined under  a  low-power  lens,  are  seen  to  be  spherical  or  lemon-shaped, 

FIG.  102  FIG.  103 

«-,*• 


"*•* 


Micrococcus  tetragenus.  Micrococcus  tetragenus  in  peritoneal 

X  1000  diameters.  fluid.    (After  Zettnow.) 

grayish-yellow  disks,  with  a  finely  granular  or  mulberry-like  surface, 
and  a  uniform  but  somewhat  roughly  dentated  border.  When  the  deep 
colonies  push  forward  to  the  surface  of  the  gelatin  they  form  white, 
elevated,  drop-like  masses,  having  a  diameter  of  1  to  2  mm.  In  gelatin 
stick  cultures  the  colonies  may  be  either  isolated  or  confluent,  in  the 
case  forming  a  thick,  white,  slimy  mass,  filling  out  the  fissures  and 
hollow  spaces  all  along  the  line  of  puncture;  on  the  surface  a  broad, 
thick  layer  of  4  to  5  mm.  in  extent  is  apparent.  The  gelatin  is  not 
liquefied. 

Growth  on  Agar  and  Blood  Serum.  —  The  colonies  appear  as  small, 
transparent,  round  points,  which  have  a  grayish-yellow  color  and  are 
slightly  elevated  above  the  surface  of  the  medium. 

Pathogenesis.  —  Subcutaneous  injections  of  a  culture  of  this  micro- 
coccus  in  minute  quantity  is  usually  fatal  to  white  mice.  The  micro- 
cocci  are  found  in  comparatively  small  numbers  in  the  blood  of  the 
vessels  and  heart,  but  are  more  numerous  in  the  spleen,  lungs,  liver, 


PRODUCERS  OF  ABSCESSES,  CELLULITIS  AND  SEPTICAEMIA     337 

and  kidneys.  Intraperitoneal  injections  given  to  guinea-pigs  and  mice 
are  followed  by  purulent  peritonitis,  beautifully  formed  cocci  in  groups 
of  four  being  obtained  in  immense  numbers  from  the  exudate.  Rabbits 
and  dogs  are  not  affected  by  large  doses  of  a  culture  subcutaneously  or 
intravenously  administered. 

The  serum  from  immunized  cases  has  not  been  used  therapeutically 
in  human  infection. 

THE  STREPTOCOCCI. 

Under  this  name  must  be  included  not  only  the  streptococci  which 
excite  inflammation  in  man,  but  all  spherical  bacteria  which  divide,  as 
a  rule,  in  one  plane  only  and  hold  cocci  together  in  greater  or  lesser 
chains.  This  name  comprises  by  no  means  so  many  varieties  of  bac- 
teria as  are  grouped  under  the  title  bacilli.  There  are,  nevertheless,  a 
considerable  number  of  distinct  groups  of  streptococci  which  differ 
decidedly  both  in  their  cultural  characteristics  and  their  pathogenic 
properties.  The  streptococci  average  about  1«  in  diameter.  None 
of  them  form  spores  or  are  motile.  They  are  rather  easily  killed  by 
disinfectants.  Those  that  are  pathogenic  develop  wholly  or  almost 
so  in  or  on  the  bodies  of  man  and  animals. 

Streptococcus  Pyogenes. — The  group  of  streptococci  which  in  its 
importance  as  related  to  human  infections  outweighs  all  other  strepto- 
cocci, is  that  which  comprises  the  streptococci  which  excite  erysipelas, 
many  cases  of  cellulitis,  abscess,  septicaemia,  pneumonia,  etc.,  and 
passes  under  the  name  of  streptococcus  pyogenes. 

This  organism  was  first  discovered  by  Koch  in  stained  sections 
of  tissue,  attacked  by  septic  processes,  and  by  Ogston  in  the  pus  of 
acute  abscesses  (1882).  It  was  obtained  by  Fehleisen  (1883)  in  pure 
cultures  from  a  case  of  erysipelas,  its  cultural  and  pathological  charac- 
ters studied  and  demonstrated  by  him  to  be  capable  of  producing 
erysipelas  in  man.  Rosenbach  (1884)  and  Krause  and  Passet  (1885) 
isolated  the  streptococcus  from  the  pus  of  acute  abscesses  and  gave  it 
the  name  of  streptococcus  pyogenes.  It  has  since  been  proved  to  be 
one  of  the  chief  etiological  factors  in  the  production  of  many  suppu- 
rative  inflammations.  Formerly  the  streptococci  of  erysipelas,  acute 
abscesses,  septicaemia,  puerperal  fever,  etc.,  were  thought  to  belong 
to  different  species,  because  they  were  observed  to  possess  apparent 
differences  in  their  biological  and  pathological  characteristics,  accord- 
ing to  the  source  from  which  they  were  obtained.  Thus  one  species  of 
streptococcus  was  believed  to  be  capable  of  causing  erysipelas  only, 
another  only  acute  abscesses,  another  sepsis,  etc.,  but  it  is  now  known 
that  the  slight  differences  between  the  majority  of  these  streptococci 
are  but  acquired  variations  of  organisms  derived  from  the  same  species 
which  are  not  permanent. 

Morphology. — The  cocci,  when  fully  developed  are  spherical  or  oval. 
They  have  no  flagella  or  spores.  They  vary  from  0.4//  to  l/i  in  diameter. 
They  van-  in  dimensions  in  different  cultures  and  even  in  different 

22 


338 


BACTERIA  PATHOGENIC  TO  MAN 


parts  of  a  single  colony.  They  multiply  by  binary  division  in  one 
direction  only,  forming  chains  of  eight,  ten,  twenty,  and  more  ele- 
ments, being,  however,  often  associated  distinctly  in  pairs.  On  solid 
media  the  cocci  occur  frequently  as  diplococci,  but  usually  they 
grow  in  longer  or  shorter  chains.  Certain  cocci  frequently  exceed 
their  fellows  greatly  in  size,  especially  in  old  cultures,  when  this  may 


FIG.  104 


FIG.  105 


Streptococci  in  peritoneal  fluid,  partly  enclosed 
in  leukocytes.    X  1000  diameters. 


Streptococci  in  throat  exudate  smeared  o» 
cover-glass.    X  1000  diameters. 


be  considered  to  be  the  result  of  involution  forms.  Some  varieties 
have  distinct  capsules  when  growing  in  the  blood  and  in  blood-serum, 
media  (Hiss). 


FIG.  106 


FIG.  107 


• 


Streptococci  from  solidified  serum  culture  appear- 
ing mostly  in  diplococci.    X  1000  diameters. 


Streptococcus  growing  in  long  chains  ii> 
bouillon  culture.    X  1000  diameters. 


Staining. — They  stain  readily  by  aniline  colors  and  the  pyogenic 
varieties  positively  by  Gram's  method.  Some  varieties,  mostly  sapro- 
phytic,  growing  in  short  chains  are  negative  to  Gram's  stain. 

Biology. — Streptococci  grow  readily  in  various  liquid  and  solid  cul- 
ture media.  The  most  favorable  temperature  for  their  development 
is  from  30°  to  37°  C.,  but  they  multiply  rather  freely  at  ordinary  room- 


P,Y"/;rr/;/Y.s  o/-'  ABSCESSES,  CLLIA' I.ITIS  AM)  SEPTIC  /-/W/.l      339 

temperature — 18°  to  20°  C.     Tlrey  are  facultative  anaerobes,  growing 
both  in  the  presence  and  absence  of  oxygen. 

Cultivation.  GROWTH  o\  (ii.i.niv. — Tubes  of  gelatin  which  ha\v 
been  inoculated  with  streptococci  by  puncture  with  the  platinum  needle 
show  on  the  surface  no  growth  beyond  the  point  of  entrance.  In  the 
depth  of  the  gelatin  on  the  second  or  third  day  a  distinct,  tiny  band 
appears,  with  granular  edges  or  made  up  of  granules.  These  granules 
may  be  very  fine  or  fairly  coarse.  They  are  nearly  translucent,  with  a 
whitish,  yellowish,  or  brownish  tinge.  With  characteristic  cultures  the 
gelatin  is  not  liquefied,  though  occasionally,  with  saprophytic  varieties, 
a  certain  amount  of  liquefaction  has  been  observed  to  take  place. 

GROWTH  ox  AGAR. — On  agar  plates  the  colonies  are  visible  after 
twelve  to  thirty  hours'  growth  at  37°  C.,  and  present  a  beautiful  appear- 
ance when  magnified  sufficiently  to  see  the  individual  cocci  in  the 
chain.  The  colonies  are  small,  not  averaging  over  0.5  mm.  in  diameter 
(pin-head).  From  different  sources  they  vary  in  size,  thickness,  mottling, 
color,  and  in  the  appearance  of  their  borders.  The  streptococcus 
growing  in  short  chains  in  bouillon  shows  but  little  tendency  to  form 
true  loops,  but  rather  projecting  rows  at  the  edges  of  the  colonies,  while 
those  growing  in  long  chains  show  beautiful  loops,  which  are  character- 
istic of  this  organism.  The  colonies  are  nearly  circular  in  shape  when 
thinly  scattered  over  the  plates,  but  irregular  in  form  when  crowded 
together. 

GROWTH  IN  BOUILLON. — Most  streptococci  grow  well  in  slightly  alka- 
line bouillon  at  37°  C.,  reaching  their  full  development  within  thirty-six 
to  forty-eight  hours.  Those  which  grow  in  long  chains  usually  give 
an  abundant  flocculent  deposit  and  leave  the  liquid  clear.  The  deposit 
may  be  in  grains,  in  tiny  flocculi,  in  larger  flakes,  or  in  tough,  almost 
membranous  masses,  the  differences  depending  on  the  strength  of  union 
between  the  pairs  of  cocci  in  the  chains.  Some  of  the  streptococci 
growing  in  long  chains,  however,  cause  the  broth  to  become  cloudy. 
This  cloudiness  may  be  only  temporary  or  it  may  be  lasting.  Those 
growing  in  short  chains,  as  a  rule,  cloud  the  broth,  this  cloudiness 
remaining  for  days  or  weeks.  A  granular  deposit  appears  at  the 
bottom  of  the  tube.  An  addition  of  0.5  to  1  per  cent,  glucose  aids  the 
development  of  streptococci,  but  the  acid  produced  tends  later  to  hasten 
their  death  and  make  them  lose  virulence.  A  trace  of  calcium  aids 
the  growth.  This  is  best  added  as  a  piece  of  marble,  which  has  tin- 
additional  advantage  of  neutralizing  some  of  the  acids  produced. 

GROWTH  IN  ASCITIC  OR  SERU.M  BOUILLON. — The  development  in  this, 
which  is  the  test  medium  for  the  growth  of  the  streptococcus,  is  more 
abundant  than  in  plain  bouillon.  The  liquid  is  clouded,  and  a  precipitate 
only  occurs  after  some  days,  the  fluid  gradually  clearing.  The  addition 
of  blood  serum  frequently  causes  streptococci,  growing  in  short  chains 
in  nutrient  bouillon,  to  produce  long  chains.  The  reverse  is  also  true, 
and  in  the  blood  all  forms  are  usually  found,  partly,  at  least,  as  diplo- 
cocci  or  in  short  chains. 

EFFECT  ON  IM  LIN. — This  is  not  fermented  by  most  varieties 


340  BACTERIA  PATHOGENIC  TO  MAN 

GROWTH  ON  SOLIDIFIED  BLOOD  SERUM. — This  is  also  an  excellent 
medium  for  the  streptococcus.  Tiny,  grayish  colonies  appear  twelve 
to  eighteen  hours  after  inoculation. 

GROWTH  IN  MILK. — All  streptococci  grow  well  in  milk.  As  a  rule, 
when  growth  is  luxuriant  a  marked  production  of  lactic  acid  with 
coagulation  of  the  casein  occurs. 

DEVELOPMENT  IN  BLOOD  AGAR. — Most  streptococci  produce  abund- 
ant hsemolytic  substances.  This  is  especially  true  of  those  from  human 
septic  infections.  As  the  pneumococci  and  other  types  of  streptococci 
produce  them  in  a  much  less  degree,  blood-agar  plates  are  a  very  useful 
means  for  a  probable  identification.  According  to  Rosenow  if  1  c.c. 
of  fresh  or  defibrinated  blood  is  added  to  6  c.c.  of  melted  agar  at 
40°  to  45°  C.,  well  shaken,  inoculated  with  the  organisms  and  poured  in 
a  Petri  dish  there  will  appear  in  twelve  to  twenty-four  hours,  if  char- 
acteristic streptococci  are  present,  tiny  colonies  surrounded  by  clear 
zones  of  about  i  to  J  inch  in  diameter.  Pneumococci  and  some 
varieties  of  streptococci  on  the  other  hand  produce  only  narrow  zones, 
but  instead  a  green  pigment. 

DURATION  OF  LIFE  OUTSIDE  OF  THE  BODY. — This  is  not,  as  a  rule, 
very  great.  When  dried  in  blood  or  pus,  however,  they  may  live  for 
several  months  at  room- temperature,  and  longer  in  an  ice-chest,  and 
in  gelatin  and  agar  cultures  they  live  for  from  one  week  to  three  months. 
In  order  to  keep  streptococci  alive  and  virulent,  it  is  best  to  transplant 
them  frequently  and  to  keep  them  in  serum  or  ascitic  fluid  bouillon  in 
small,  sealed  glass  tubes  in  the  ice-chest. 

RESISTANCE  TO  HEAT  AND  CHEMICALS. — The  thermal  death  point  of 
the  streptococcus  is  between  52°  and  54°  C.,  the  time  of  exposure  being 
ten  or  twenty  minutes. 

Mercuric  chloride,  1:2500;  sulphate  of  copper,  1:200;  trichloride  of 
iodine,  1:750;  peroxide  of  hydrogen,  1:50;  carbolic  acid,  1:300; 
<cresol,  1:250;  lysol,  1:300;  creolin,  1:130,  all  kill  streptococci  within 
a  few  minutes. 

Pathogenesis. — The  majority  of  test  animals  are  not  very  susceptible 
to  infection  by  the  streptococcus,  and  hence  it  is  difficult  to  obtain  any 
definite  pathological  alterations  in  their  tissues  through  the  inoculation 
into  them  of  cultures  of  this  organism  by  any  of  the  methods  ordinarily 
practised.  White  mice  and  rabbits,  under  similar  conditions,  are  the 
most  susceptible,  and  these  animals  are,  therefore,  usually  employed 
for  experimentation.  Streptococci,  however,  differ  greatly  in  the  effects 
•which  they  produce  in  inoculated  animals,  according  to  their  animal 
virulence,  which  is  very  different  from  human  virulence.  The  most 
virulent  when  injected  in  the  minutest  quantity  into  the  circulation  or 
into  the  subcutaneous  tissues  of  a  mouse  or  rabbit,  produce  death  by 
septicaemia.  Those  of  somewhat  less  virulence  produce  the  same  result 
when  injected  in  considerable  quantities.  Those  still  less  pathogenic 
produce  septicaemia,  which  may  be  mild  or  severe,  when  injected  into 
the  circulation;  but  when  injected  subcutaneously,  they  produce  abscess 
or  erysipelas.  The  remaining  streptococci,  unless  introduced  in  quan- 


PRODUCERS  Of  .\ntCESSES,  CELLULITIS  AXD  SEPTICAEMIA     341 

titles  of  20  c.c.  or  over,  produce  only  a  slight  redness,  or  no  reaction  at 
all,  when  injected  subcutaneously,  and  little  or  no  effect  when  injected 
directly  into  the  circulation.  Many  of  the  streptococci  obtained  from  cases 
of  cellulitis,  abscess,  empyema,  and  septicaemia  belong  to  this  group. 

A  number  of  varieties  of  streptococci  have  thus  been  discovered,, 
differing  in  virulence  and  in  their  growth  on  artificial  media;  but  all 
attempts  to  separate  them  into  various  classes,  even  with  the  use  of 
specific  serum,  have  largely  failed,  because  the  differences  observed, 
though  often  marked,  are  not  constant,  many  varieties  having  been 
found  to  lose  their  distinctive  characteristics,  and  even  to  apparently 
change  from  one  class  to  another.  A  further  objection  to  any  of  the 
existing  classification  of  streptococci,  which  are  based  on  the  manner 
of  growth  on  artificial  culture  media,  is  that  it  has  been  impossible  to- 
make  any  which  would  at  the  same  time  give  even  an  approximate 
idea  of  their  virulence.  Experiments  have  proved  that  streptococci 
originally  virulent  may  become  non-virulent  after  long  cultivation  on 
artificial  media,  and,  again,  that  they  may  return  to  their  original 
properties  after  being  passed  through  the  bodies  of  susceptible  animals. 
The  peculiar  type  of  virulence  which  they  may  acquire  tends  to  per- 
petuate itself,  at  least  for  a  considerable  time. 

One  important  fact  that  experience  teaches  us  is,  that  those  streptococci 
which  are  the  most  dangerous  are  those  which  have  come  immediately 
from  septic  conditions,  and  the  more  virulent  the  case  the  more  virulent 
the  streptococci  are  apt  to  be  for  animals  of  the  same  species.  There 
seems  also  to  be  a  strong  tendency  for  a  streptococcus  to  produce  the 
same  inflammation,  when  inoculated,  as  the  one  from  which  it  was 
obtained;  for  example,  streptococci  from  erysipelas  tend  to  produce 
erysipelas,  from  septicaemia  to  produce  septicaemia,  etc.  Streptococci, 
however,  obtained  from  different  sources  (abscesses,  puerperal  fever, 
sepsis,  erysipelas,  etc.)  are  in  many  instances  capable,  under  favorable 
conditions,  of  producing  erysipelas  when  inoculated  into  the  ear  of  a 
rabbit,  as  has  been  proved  by  experiment,  provided  they  possess 
sufficient  virulence. 

OCCURRENCE  ix  MAN. — Streptococci  have  been  found  in  man  as  the 
primary  cause  of  infection  in  the  following  diseases :  Erysipelas,  circum- 
scribed and  extensive  acute  abscesses,  impetigo,  cellulitis  (circumscrilxMl 
as  well  as  diffused),  sepsis,  puerperal  infection,  lymphatic  abscesses, 
angina,  bronchopneumonia,  periostitis,  osteomyelitis,  synovitis,  otitis 
media,  mastoiditis,  meningitis,  pleurisy,  empyema,  and  endocarditis. 
Associated  with  other  bacteria  in  diseases  of  which  they  were  the  specific 
cause,  they  have  also  been  found  as  the  secondary  or  mixed  infection  in 
many  diseases,  such  as  in  pulmonary  tuberculosis,  bronchopneumonia, 
septic  diphtheria,  and  diphtheritic  scarlatina.  In  diphtheritic  false 
membranes  this  micrococcus  is  very  commonly  present,  and  is  frequently 
the  source  of  deeper  infection,  such  as  abscesses  and  septicaemia; 'and 
in  certain  cases  attended  with  a  diphtheritic  exudation,  in  which  the 
Loeffler  bacillus  has  not  been  found  by  competent  bacteriologists,  it 
seems  probable  that  the  streptococcus  pyogenes,  alone  or  with  other 


342  BACTERIA  PATHOGENIC  TO  MAN 

pyogenic  cocci,  is  responsible  for  the  local  inflammation  and  its  results. 
These  forms  of  so-called  diphtheria,  as  first  pointed  out  by  Prudden, 
are  most  commonly  associated  with  scarlatina  and  measles,  erysipelas, 
and  phlegmonous  inflammation,  or  occur  in  individuals  exposed  to  these 
or  other  infectious  diseases.  So  uniformly  are  streptococci  present  in 
the  pseudomembranous  inflammation  of  patients  sick  with  scarlet 
fever,  that  many  investigators  have  suspected  a  special  variety  of  them 
to  be  the  cause  of  this  disease.  The  same  is  true  for  smallpox.  Many 
varieties  are  regularly  found,  however,  in  the  throat  secretion  of  healthy 
individuals  (in  100  examinations  by  us  we  found  them  in  83,  and  probably 
could  have  found  them  in  some  of  the  others  by  longer  search).  Their 
presence  in  scarlet  fever  and  smallpox  is  most  probably  due  to  their 
increase  in  the  disordered  mucous  membrane  and  entrance  into  the  cir- 
culation when  the  protective  properties  of  the  blood  have  been  lowered. 

The  causal  relation  of  the  streptococcus  to  the  above-mentioned 
diseases  has  been  amply  proved  by  inoculation  experiments  both  in  man 
and  animals.  Fehleisen  has  inoculated  cultures,  obtained  in  the  first 
instance  from  the  skin  of  patients  with  erysipelas,  into  patients  in  the 
hospital  suffering  from  inoperable  malignant  growths — lupus,  carcinoma, 
and  sarcoma — and  has  obtained  positive  results,  a  typical  erysipelatous 
inflammation  having  developed  around  the  point  of  inoculation  after  a 
period  of  incubation  of  from  fifteen  to  sixty  hours.  This  was  attended 
with  chilly  sensations  and  an  elevation  of  temperature.  Persons  who 
had  recently  recovered  from  an  attack  of  erysipelas  proved  to  be  immune. 
These  experiments  were  undertaken  on  the  ground  that  malignant 
tumors  had  previously  been  found  to  improve  or  entirely  disappear  in 
persons  who  had  recovered  from  accidental  erysipelas.  During  the  last 
few  years  this  fact  has  been  therapeutically  applied  to  the  treatment  of 
malignant  tumors  by  the  artificial  production  of  erysipelas  by  the 
inoculation  of  pure  cultures  of  streptococcus  or  of  their  toxic  products. 

RESULTS  FROM  INJECTIONS  IN  TUMORS. — In  some  cases  of  sarcoma 
this  method  has  met  with  considerable  success;  in  carcinoma,  however, 
the  results  have  been  very  slight.  In  this  country  the  experimental  work 
upon  this  subject  and  the  actual  treatment  of  cases  have  been  largely 
carried  out  by  or  under  the  direction  of  Coley.  He  has  kindly  sent  me 
the  following  notes  on  his  results: 

"The  improvement  and  inhibitory  action  which  the  toxins  have  upon 
carcinoma  have  proved  to  be,  in  nearly  all  cases,  but  temporary. 

"  On  the  other  hand,  in  sarcoma,  which  is  the  only  form  of  malignant 
tumor  in  which  I  have  advocated  the  treatment,  sufficient  time  has 
elapsed  to  enable  us  to  draw  the  following  conclusions : 

"The  toxins  injected  subcutaneously  into  the  tissues,  either  into  the 
tumor  substance  or  into  parts  remote  from  the  tumor,  exercise  a  distinctly 
inhibitory  action  upon  the  growth  of  nearly  all  varieties  of  sarcoma. 
This  action  is  the  least  marked  in  melanotic  sarcoma,  and  thus  far  no 
cases  of  this  form  of  tumor  have  disappeared  under  the  treatment.  The 
influence  of  the  toxins  upon  round-celled  sarcoma  is  much  more  powerful 
than  it  is  upon  melanotic,  although  distinctly  less  than  upon  the  spindle- 


OF  ABSCESSES,  CELLULITIS  AND  SEPTICAEMIA     343 

celled  variety.  A  number  of  cases  of  round-celled  sarcoma  in  which  the 
diagnosis  was  unquestioned  disappeared,  and  the  patients  have  remained 
\\ell  beyond  three  years.  Nearly  half  of  the  cases  treated  showed  no 
appreciable  decrease  in  si/.e;  the  majority  of  the  others  which  did  show 
marked  improvement  at  first,  after  decreasing  in  size  for  a  few  weeks, 
again  began  to  increase  and  were  no  longer  influenced  by  the  treatment. 

"  In  half  of  the  cases  of  spindle-celled  sarcoma  treated  by  the  toxins  the 
disease  had  disappeared  entirely,  and  the  majority  of  the  successful  cases 
have  remained  well  sufficiently  long  to  justify  their  being  regarded  as  cured. 
It  should  be  distinctly  stated  that  all  of  the  tumors  under  consideration 
were  inoperable,  as  I  have  never  advised  treatment  except  in  such  cases. 

"I  have  now  a  number  of  cases  of  spindle-celled  sarcoma  which 
have  remained  well  beyond  three  years;  one  case  of  mixed  (round  and 
spindle)  celled,  after  remaining  well  three  years,  had  a  return  in  the 
abdomen,  and  died  about  eight  months  later.  The  result  here  certainly 
establishes  the  correctness  of  the  early  diagnosis." 

PRODUCTION  OF  Toxic  SUBSTANCES. — There  is  no  doubt  that  the 
streptococcus  causes  fever,  general  symptoms  of  intoxication,  and  death 
by  means  of  toxic  substances  which  it  forms  in  its  growth;  but  we 
know  but  little  about  these  substances  or  how  they  are  produced.  The 
poisons  while  partly  extracellular  are  mostly  contained  in  the  cell  sub- 
stance. Heat  destroys  a  portion  of  them.  They  appear  to  attack 
especially  the  red  blood  cells,  and  this  ha?molytic  action  seems  to  be  to 
some  degree  in  proportion  to  the  virulence  of  the  organism. 

SUSCEPTIBILITY  TO  STREPTOCOCCUS  INFECTION. — As  with  the  ever- 
present  staphylococci,  whose  virulence,  as  we  have  seen,  is  usually 
slight,  the  streptococci  are  more  likely  to  invade  the  tissues,  forming 
abscesses  or  erysipelatous  and  phlegmonous  inflammation  in  man 
when  the  standard  of  health  is  reduced  from  any  cause,  and  espe- 
•cially  when  by  absorption  or  retention  various  toxic  organic  prod- 
ucts are  present  in  the  body  in  excess.  It  is  thus  that  the  liability  to 
these  local  infections,  as  complications  of  operations  or  sequela?  of 
various  specific  infectious  diseases,  in  the  victims  of  chronic  alcoholism, 
and  constitutional  affections,  etc.,  are  to  be  explained.  It  seems  estab- 
lished that  the' absorption  of  toxic  products  formed  in  the  alimentary 
canal  as  a  result  of  the  ingestion  of  improper  food,  or  in  consequence 
of  abnormal  fermentative  changes  in  the  contents  of  the  intestine,  or 
from  constipation,  predispose  to  infection. 

Immunity. — Knorr  succeeded  in  producing  a  moderate  immunity 
in  rabbits  against  an  intensely  virulent  streptococcus  by  injections  of 
very  slightly  virulent  cultures.  Pasquale  was  able  to  immunize  these 
animals  partially  against  septica?mia.  Marmorek  has  immunized 
sheep,  asses,  and  horses  against  very  large  doses  of  a  streptococcus, 
which  though  but  slightly  virulent  for  them  was  intensely  so  for  rabbits. 

In  none  of  the  streptococcus  inflammations  do  we  notice  much  appar- 
ent tendency  to  the  production  of  immunizing  and  curative  substances 
in  the  blood  by  a  single  infection. 

Severe   general   infections   usually   progress   to   a   fatal   termination 


344 


BACTERIA  PATHOGENIC  TO  MAN 


after  a  few  days,  weeks,  or  months.  It  is  true,  however,  that  cases  of 
erysipelas,  cellulitis,  and  abscess,  after  periods  varying  from  a  few  days 
to  months,  tend  to  recover,  and  to  a  certain  extent,  therefore,  we  may 
assume  that  protective  agents  have  been  produced.  In  these  cases,  how- 
ever, we  know  from  experience  that  faulty  treatment,  by  lessening  the 
local  or  general  resistance,  would,  as  a  rule,  cause  the  subsiding  infection 
to  again  progress  perhaps  even  to  a  more  serious  extent  than  the  original 
attack.  Koch  and  Petruschky  tried  a  most  interesting  experiment.  They 
inoculated  cutaneously  a  man  suffering  from  a  malignant  tumor  with  a 
streptococcus  obtained  from  erysipelas.  He  developed  a  moderately 
severe  attack,  which  lasted  about  ten  days.  On  its  subsidence  they  re- 
inoculated  him;  a  new  attack  developed,  which  ran  the  same  course  and 
over  the  same  area.  This  was  repeated  ten  times  with  the  same  results. 

This  experiment  proved  that  in  this  case,  at  least,  little  if  any  lasting 
curative  or  immunizing  substances  were  produced  by  repeated  attacks 
of  erysipelas,  and  that  the  recovery  from  each  attack  was  due  to  local 
and  transitory  protective  developments. 

The  severe  forms  of  infection,  such  as  septicaemia  following  injuries, 
operations,  and  puerperal  infections,  shoAv  little  tendency  to  be  arrested 
after  being  well  established.  Having,  then,  in  remembrance,  the  above 
facts,  let  us  consider  the  results  already  obtained  in  the  experimental 
immunization  and  treatment  of  animals  and  men  suffering  from  or  in 
danger  of  infection  with  streptococci.  One  method  is  now  chiefly  used 
for  the  immunization  and  attempt  to  produce  curative  substances  in 
animals,  namely  the  injection  in  gradually  increasing  doses  of  the  living, 
virulent  streptococcus  itself.  Marmorek  was  the  first  to  attempt  the 
production  of  a  curative  serum  on  a  large  scale. 

Influence  of  Serum  from  Immunized  Animals  upon  Streptococcus  Infec- 
tions in  Other  Animals. — In  the  table  are  given  the  results  following  the 
injection  of  small  amounts  of  a  serum  which  represents  in  immunizing 
value  what  about  one-third  of  the  horses  are  able  to  produce.  In  the 
following  experiments  the  serum  and  culture  were  injected  subcuta- 
neously  in  rabbits  at  the  same  time,  but  in  opposite  sides  of  the  body : 

TABLE  Showing  Strength^of  Average  Grade  of  Antistreptococcic  Serum  given  by  Selected 
Horses  after  six  months  of  Injection  of  suitabe  amounts  of  Living  Streptococci. 


Weight 
of 
rabbit. 

Amounts 
inoculated. 

Result. 

Autopsy. 

Serum  and  culture  : 

Grms. 

Serum.      Cult. 

1.  Inoculated  together  .... 

1430 

0.25  c.c.    0.01  c.c. 

Lived 

2.          "               " 

1350 

0.125"      0.01   " 

« 

3.  On  opposite  sides      .... 

1770 

0.1      "      0.01   " 

" 

4.           "                " 

1630 

0.1      "      0.01   " 

" 

Controls  : 

1.  Rabbits  injected  with  culture  only 
2.       « 

1750 
1870 

0.001" 
0.001" 

Died  in 
4  days. 
Died  in 
24  hrs. 

Streptococcic 
infection. 
Streptococcic 
infection. 

PRODUCERS  OP  ABSCESSES,  CELLULITIS  AXD  SEPTICAEMIA 


The  above  results  have  been  repeatedly  obtained,  and  are  absolutely 
conclusive  that  the  serum  of  properly  selected  animals,  which  have 
been  repeatedly  injected  with  living  streptococci  in  suitable  doses, 
possesses  bactericidal  properties  upon  the  same  streptococcus  when  it 
comes  in  contact  with  it  within  the  bodies  of  animals. 

Definite  protection  from  the  serum  has  been  obtained  by  many 
reliable  observers  since  Marrnorek's  first  reports. 

Is  Protection  Afforded  by  the  Same  Serum  against  all  Varieties  of  Strep- 
tococci? —  We  have  tested  the  protective  value  of  one  serum  against 
streptococci  derived  from  five  different  sources.  First,  the  streptococcus 
Driven  us  by  Marmorek,  which  was  obtained  from  a  case  of  angina. 
Its  virulence  is  now  such,  after  having  passed  through  hundreds  Jof 
rabbits,  that  0.000001  c.c.  is  the  average  fatal  dose.  Second,  a  strepto- 
coccus obtained  from  a  case  of  erysipelas  in  England.  Its  virulence 
is  0.00001  c.c.  on  the  average.  Third,  a  streptococcus  obtained  from 
a  case  of  cellulitis,  its  virulence  being  about  6  c.c.  Fourth,  a  strepto- 
coccus sent.  me  by  Theobald  Smith.  Its  virulence  is  such  that  0.1  c.c. 
is  the  average  fatal  dose.  Fifth,  another  culture  sent  me  by  Smith,. 
which  grew  in  short  chains  and  was  obtained  from  milk;  its  virulence 
was  similar  to  No.  4. 

Against  the  first  three  streptococci  derived  from  three  different 
varieties  of  infection  existing  in  three  different  countries  the  serum 
produced  by  the  streptococcus  from  England  had  nearly  the  same 
value.  Against  the  latter  two  streptococci,  as  well  as  against  a  strepto- 
coccus from  a  case  of  endocarditis,  which  resembled  in  some  respects 
the  pneumococci  arid  a  pneumococcus,  the  serum  had  no  effect. 

The  results  of  numerous  investigators  indicate  that  the  majority  of 
streptococci  met  with  in  septic  infections  will  be  influenced  by  the  same 
serum.  Many  more  streptococci,  however,  must  be  obtained  from 
human  infections  and  tested  before  we  can  be  certain  of  this.  Those 
obtained  from  cases  of  pneumonia  and  endocarditis  which  have  some 
resemblance  to  pneumococci  and  which  are  not  very  virulent  in  animals,. 
are  especially  in  need  of  investigation. 

Preparation  of  the  Serum.  —  Antistreptococcus  serum  is  obtained  from 
the  horse  after  treatment  by  repeated  injections  of  living  streptococcus 
cultures  of  streptococci  derived  from  human  sources.  As  a  rule,  a 
number  of  varieties  are  given  at  the  same  time  so  that  the  serum  will 
be  active  against  any  variety  causing  the  infection.  If  the  serum  is  ta 
be  used  in  scarlet  fever,  the  streptococci  used  should  be  from  cases  of 
scarlet  fever.  The  procuring  of  a  serum  of  the  highest  potency  requires 
a  considerable  number  of  animals,  for  some  produce  with  the  same 
treatment  a  more  protective  serum  than  others.  The  serum  must  be 
sterile  from  streptococcus  as  well  as  from  other  contaminations. 

Stability  of  the  Serum.  —  Unfortunately,  after  several  weeks  or  months, 
the  serum,  as  a  rule,  loses  most  of  its  protective  value.  It  should  be 
kept  in  a  cold  and  dark  place. 

Standardization  of  the  Value  of  the  Serum.  —  The  value  of  the  serum  is 
measured  by  the  amount  required  to  protect  against  a  multiple  of  a  fatal 


346  BACTERIA  PATHOGEXIC  TO  MAX 

dose  of  a  very  virulent  streptococcus.  The  dose  is  usually  a  thousand 
times  the  average  fatal  amount  of  a  very  virulent  streptococcus. 

This  method  gives,  as  a  rule,  to  those  unfamiliar  with  bacteriology, 
an  exaggerated  idea  of  the  potency  of  the  serum. 

A  thousand  times  the  amount  of  a  very  virulent  streptococcus  cul- 
ture required  to  kill  an  animal  by  producing  septicaemia  is  more  easily 
controlled  than  four  times  a  fatal  dose  of  a  slightly  virulent  strepto- 
coccus. The  serum  acts  upon  a  certain  quantity  of  organisms,  while 
it  is  only  their  enormous  multiplication  in  the  animal  which  kills. 

It  is  entirely  different  in  case  of  an  antitoxin  which  does  not  prevent 
primarily  the  growth  of  the  germ,  but  neutralizes  a  chemical  substance 
— its  toxin. 

Therapeutic  Results. — To  estimate  the  exact  present  and  future  value 
of  antistreptococcus  serum  is  a  matter  of  the  utmost  difficulty.  Many 
of  the  cases  reported  are  of  little  or  no  help,  because  no  cultures 
having  been  made,  we  are  in  doubt  as  to  the  nature  of  the  bacterial 
infection. 

Marmorek's  results  are  by  far  the  best  reported,  but  without  casting 
any  doubt  upon  the  justification  of  his  conclusions,  from  the  data  at 
his  command,  I  believe  they  undoubtedly  give  too  favorable  a  view  of 
the  value  of  the  serum. 

In  the  few  cases  of  puerperal  fever,  erysipelas,  wound  infection, 
scarlet  fever,  and  bronchopneumonia  that  we  have  seen,  the  apparent 
results  under  the  treatment  have  not  been  uniform.  Only  occasionally 
have  we  seen  results  which  appeared  to  be  distinctly  due  to  the  serum. 

In  a  number  of  cases  of  septicaemia  where  for  days  chills  had 
occurred  daily  they  ceased  absolutely  or  lessened  under  daily  doses  of 
20  to  50  c.c.  The  temperature,  though  ceasing  to  rise  to  such  heights, 
did  not  average  more  than  one  or  two  degrees  lower  than  before  the 
injections.  In  some  cases  the  serum  treatment  was  kept  up  for  four 
weeks.  Some  cases  convalesced;  others  after  a  week  or  more  grew 
worse  and  died.  In  some  cases  the  temperature  fell  immediately  upon 
giving  the  first  injection  of  serum,  and  after  subsequent  injections  re- 
mained normal,  and  the  cases  seemed  greatly  benefited.  As  a  rule,  in 
these  cases  no  streptococci  or  any  other  organisms  were  obtained  from 
the  blood.  In  bronchopneumonia,  laryngeal  diphtheria,  scarlet  fever, 
smallpox,  and  phthisis,  we  have  seen  absolutely  no  effect.  In  the 
exanthemata  our  injections  were  much  smaller  than  those  used  in 
Vienna,  in  which  city  very  striking  results  are  reported  from  100  c.c. 
doses. 

The  results  obtained  here  in  New  York  by  both  physicians  and  sur- 
geons have  not,  on  the  whole,  been  very  encouraging. 

In  some  of  the  cases  where  apparently  favorable  results  were  obtained 
other  bacteria  than  streptococci  were  found  to  be  the  cause  of  the  dis- 
ease. We  believe  that  the  following  conclusions  will  be  found  fairly 
accurate : 

A  single  antistreptococcic  serum  protects  healthy  rabbits  from  infec- 
tion from  most  of  the  streptococci  obtained  from  human  sepsis,  but 


PRODUCERS  OF  AHSCKSSES,  CKLl.l' 1.ITIS  AND  si-'.rTH'.KM  1 A     347 

not  from  all.  Failure  to  do  good  in  human  infection  cannot,  as  a  rule, 
he  attributed  to  the  variety  of  streptococcus.  The  serum  will  in  animals 
limit  an  infection  already  started  if  it  has  not  progressed  too  far.  The 
apparent  therapeutic  results  in  cases  of  human  streptococcus  infection 
are  variable.  In  some  cases  the  disease  has  undoubtedly  advanced  in 
spite  of  large  injections,  and  here  it  has  not  seemed  to  have  had  any 
effect.  In  other  cases  good  observers  rightly  or  wrongly  believe  they 
have  noticed  great  improvement  from  it.  Except  rashes,  few  have 
noticed  deleterious  results,  although  very  large  doses  have  been  followed 
in  several  instances,  for  a  short  time,  by  albuminous  urine  and  even 
temporary  suppression. 

In  suitable  cases  we  are  warranted,  we  believe,  in  trying  it,  but  we 
should  not  expect  very  striking  results. 

For  our  own  satisfaction,  and  to  increase  our  knowledge,  we  should 
always  have  satisfactory  cultures  made  when  possible,  and  the  strepto- 
cocci, if  obtained,  tested  with  the  serum  used  in  the  treatment.  In  the 
cases  where  we  want  most  to  use  the  serum,  such  as  puerperal  fever, 
septicaemia,  ulcerative  endocarditis,  etc.,  we  find  that  it  is  very  difficult 
to  make  a  bacteriological  diagnosis  from  the  symptoms,  and  in  over 
one-half  of  the  cases  even  the  bacteriological  examination  carried  out 
in  the  most  thorough  way  will  fail  to  detect  the  special  variety  of  bac- 
teria causing  the  infection.  This  is  often  a  great  hindrance  to  the 
proper  use  of  curative  antistreptococcic  serum,  for  it,  of  course,  has 
no  specific  effect  upon  the  course  of  any  infection  except  that  due  to 
the  streptococcus  and  the  full  effect  only  on  its  own  type. 

Care  should  be  taken  to  get  only  recently  tested  serum,  for  after  six 
weeks  the  best  serum  is  almost  inert;  much  on  the  market  is  worthless, 
and  as  it  is  weak,  and  the  testing  for  strength  is  still  very  crude,  full 
doses  (10  to  20  c.c.)  of  serum  should  be  given  if  the  case  is  at  all  serious, 
for  the  dose  is  limited  only  by  the  amount  of  horse  serum  which  we 
feel  it  safe  to  give,  not  because  we  have  sufficient  protective  substance. 

Bacteriological  Diagnosis. 

Streptococci,  using  the  name  in  a  broad  sense,  can  often  be  demon- 
strated microscopically  by  simply  making  a  smear  preparation  oj 
the  suspected  material  and  staining  with  methylene-blue  solution  or 
diluted  Ziehl's  fluid.  In  order  to  demonstrate  them  microscopically  in 
the  tissues,  the  sections  are  best  stained  by  Kiihne's  methylene-blue 
method.  In  all  cases,  even  when  the  microscopic  examination  fails, 
the  cocci  may  be  found  by  the  culture  method  on  plate  agar  or  slanted 
agar  at  37°  C.  To  obtain  them  from  a  case  of  erysipelas  it  is  best 
to  excise  a  small  piece  of  skin  from  the  margin  of  the  erysipelatous 
area  in  which  the  cocci  are  most  numerous;  this  is  crushed  up  and  part 
-of  it  transferred  to  ascitic  or  serum  bouillon,  and  part  is  streaked 
across  freshly  solidified  agar  in  a  Petri  dish  on  which  a  drop  of  sterile 
rabbit's  blood  had  previously  been  placed.  Both  are  kept  in  the  incu- 
bator at  37°  C. 


348  BACTERIA  PATHOGENIC  TO  MAN 

In  septicaemia  the  culture  method  is  always  required  to  demonstrate 
the  presence  of  streptococci,  as  the  microscopic  examination  of  speci- 
mens of  blood  is  not  sufficient.  For  this  purpose  from  10  to  15  c.c.  of 
the  blood  should  be  drawn  from  the  vein  of  the  arm  aseptically  by 
means  of  a  hypodermic  needle,  and  to  each  of  three  tubes  containing 
10  c.c.  of  melted  nutrient  agar  kept  at  about  43°  C.  1  c.c.  of  blood  is 
added.  After  thoroughly  mixing  the  contents  are  poured  into  Petri 
dishes.  The  remainder  is  added  to  flasks  or  tubes  of  nutrient  broth,, 
in  order  to  produce  an  adequate  development  of  the  cocci,  which  are 
found  in  small  numbers  in  the  bloodvessels.  Petruschky  is  of  the 
opinion  that  the  cocci  can  best  be  shown  in  blood  by  animal  inocu- 
lation. Having  withdrawn  from  the  patient  10  c.c.  of  blood  by  means 
of  a  hypodermic  syringe,  under  aseptic  precautions,  he  injects  a  por- 
tion of  this  into  the  abdominal  cavity  of  a  mouse,  while  the  other  por- 
tion is  planted  in  bouillon.  Mice  thus  inoculated  die  from  septicaemia 
when  virulent  streptococci  are  present  only  in  very  small  numbers  in 
the  blood.  If  a  successful  inoculation  takes  place  we  can,  through  the 
absence  or  presence  of  the  development  of  capsules,  often  differentiate 
between  the  pneumococcus  and  the  streptococcus,  which  cultures  may 
fail  to  do.  The  development  of  a  wide,  clear  zone  about  the  colonies,, 
without  a  development  of  green  pigment,  indicates  that  the  streptococci 
belong  to  the  pyogenes  type.  The  absence  of  a  definite  zone  and  the 
development  of  a  green  color  indicates  that  they  are  pneumococci  or 
streptococci  which  in  these  two  respects  resemble  pneumococci.  The 
growth  in  the  Hiss  inulin  serum  medium  will  generally  differentiate 
between  the  two,  as  the  pneumococci  usually  coagulate  the  serum, 
while  the  great  majority  of  streptococci  do  not.,  The  morphological 
and  cultural  characteristics  of  the  streptococcus  give  us,  unfortunately  r 
no  absolute  knowledge  as  to  the  influence  which  the  protecting  serum 
will  have.  The  actual  test  is  here  our  only  method.  The  detection  of 
the  streptococcus  in  the  blood  is  in  itself  an  unfavorable  prognostic 
sign. 

The  blood  cultures  in  many  cases  of  septicaemia  give  no  positive 
results,  for  many  of  these  cases  develop  their  symptoms  and  even  die 
from  the  absorption  of  toxins  from  the  local  infection,  such  as  an  ampu- 
tation wound  or  an  infected  uterus  or  peritoneum,  and  the  bacteria 
never  invade  the  blood.  When  we  get  negative  results  we  are,  as  a  rule, 
utterly  unable  to  test  the  case  with  curative  serums  with  any  accuracy, 
for  the  sepsis  may  be  due  to  either  the  streptococcus,  colon  bacillus, 
staphylococcus,  or  a  number  of  other  pathogenic  varieties  of  bacteria. 


CHAPTER  XXV. 

THE  DIPLOCOCCUS  OF  PNEUMONIA  (PNEUMOCOCCUS,  STREPTO- 
COCCUS LANCEOLATUS,  MICROCOCCUS  LANCEOLATUS).    THE 
PNEUMOBACILLUS  (FRIEDLANDER  BACILLUS). 

The  Diplococcus  of  Pneumonia. 

THE  diplococcus  of  pneumonia  was  observed  in  1880  almost  simul- 
taneously by  Steinberg  and  Pasteur  in  the  blood  of  rabbits  inoculated 
with  human  saliva.  In  the  next  few  years  Talamon,  Friedlander, 
A.  Fraenkel,  Weichselbaum,  and  others  subjected  this  micro-organism 
to  an  extended  series  of  investigations  and  proved  it  to  be  the  chief 
^tiological  factor  in  the  production  of  lobar  or  croupous  pneumonia 
in  man. 

The  outcome  of  the  various  investigations  proved  that  the  acute  lung 
inflammations,  especially  when  not  of  the  frank  lobar  pneumonia  type, 
are  not  excited  by  a  single  variety  of  micro-organism,  and  that  the 
bacteria  involved  in  the  production  of  pneumonias  are  also  met  with 
In  inflammations  of  other  tissues. 

In  any  individual  pneumonic  inflammation  it  is  also  found  that  more 
than  one  variety  of  bacteria  may  be  active,  either  from  the  start  or  as  a 
later  addition  to  the  original  primary  infection. 

Among  all  the  micro-organisms  active  in  exciting  pneumonia,  the 
•diplococcus  of  pneumonia  is  by  far  the  most  common,  being  almost 
always  present  in  primary  lobar  pneumonia  and  as  frequently  as  any 
other  germ  in  acute  bronchopneumonia  and  metastatic  forms.  Besides 
the  different  varieties  of  pneumococci  the  following  bacteria  are  capable  of 
•exciting  pneumonia:  streptococcus  pyogenes,  staphylococcus  pyogenes, 
bacillus  pneumonias,  bacillus  influenzas,  bacillus  pestis,  bacillus  diph- 
therias, bacillus  typhi,  bacillus  coli,  and  the  bacillus  tuberculosis.  Since 
the  varieties  of  bacteria  exciting  acute  pneumonia,  with  the  exception 
of  the  pneumococcus,  are  met  with  more  frequently  in  other  inflam- 
mations and  have  been  described  elsewhere,  they  will  only  be  noticed 
in  this  chapter  so  far  as  their  relation  to  pneumonia  demand. 

Morphology. — Typically,  the  pneumococcus  occurs  as  spherical  or  oval 
<;occi,  usually  united  in  pairs,  but  sometimes  in  longer  or  shorter  chains 
-consisting  of  from  three  to  six  or  more  elements  and  resembling  the  strep- 
tococcus. The  cells,  as  they  commonly  occur  in  pairs,  are  somewhat  oval 
in  shape,  being  usually  pointed  at  one  end — hence  the  name  lanceolatus 
or  lancet-shaped.  When  thus  united  the  junction,  as  a  rule,  is  between 
the  broad  ends  of  the  oval,  with  the  pointed  ends  turned  outward;  but 
variation  in  form  and  arrangement  .of  the  cells  is  characteristic  of  this 


350 


BACTERIA  PATHOGENIC  TO  MAN 


organism,  there  being  great  differences  according  to  the  source  from 
which  they  are  obtained.  As  observed  in  the  sputum  and  blood  it  is 
usually  in  pairs  of  lancet-shaped  elements,  which  are  surrounded  by  a 
capsule.  (See  Fig.  108.)  When  grown  in  fluid  culture  media  longer  or 
shorter  chains  are  frequently  formed,  which  can  scarcely  be  distin- 
guished from  chains  of  certain  streptococci,  except  that,  as  a  rule,  the 
length  of  the  chain  is  less  and  the  pairs  of  diplococci  are  farther  apart. 
In  cultures  the  individual  cells  are  almost  spherical  in  shape,  and 
except  in  certain  varieties  are  rarely  surrounded  by  a  capsule.  (See 
Fig.  109.)  The  pneumococcus  is  by  some  classed  as  a  streptococcus. 

The  capsule  is  best  seen  in  stained  preparations  from  the  blood  and 
exudates  of  fibrinous  pneumonia  or  from  the  blood  of  an  inoculated 
animal,  especially  the  mouse,  in  which  it  is  commonly,  though  not 


FIG.  108 


FIG.  109 


*4lr 


$."* 


Diplococcus  of  pneumonia  from  blood,  with  sur- 
rounding capsule  stained  by  method  of  Hiss. 


Pneumococcus  from  bouillon  culture, 
resembling  streptococcus. 


always,  present.  It  is  seldom  seen  in  preparations  from  cultures  unless 
special  media  are  employed.  Flagella  are  not  present. 

Staining. — It  stains  readily  with  ordinary  aniline  colors;  it  is  not 
decolorized  after  staining  by  Gram's  method.  The  capsule  may  be 
demonstrated  in  blood  or  sputum  either  by  Gram's  or  Welch's  (glacial 
acetic  acid)  method,  or  the  copper  sulphate  method  of  Hiss. 

Biology. — It  grows  equally  well  with  or  without  oxygen,  being  thus 
both  aerobic  and  facultative  anaerobic;  its  parasitic  nature  is  exhibited 
by  the  short  range  of  temperature  at  which  it  usually  grows — viz.,  from 
25°  to  42°  C.— best  at  37°  C.  In  the  cultivation  of  this  organism 
neutral  or  slightly  alkaline  media  should  be  employed.  The  organism 
grows  feebly  on  the  serum-free  culture  media  ordinarily  employed  for 
the  cultivation  of  bacteria — viz.,  on  nutrient  agar  and  gelatin,  in 
bouillon.  The  best  medium  for  its  growth  is  a  mixture  of  one-third 


mi-:  DIPLOCOCCU8  <>!•'  r\i-:r.Mn\i.\ 

human  or  ani  i  al  blood  serum  or  ascitic  or  pleuritic  fluid  and  two- 
thirds  bouillon  or  nutrient  agar  growth  in  milk. 

GROWTH  IN  M U.K. —It  grows  readily  in  milk,  causing  coagulation 
with  the  production  of  acid,  though  this  is  not  constant  with  some 
forms  intermediate  between  the  streptococcus  and  pneumococcus. 

GROWTH  ox  AGAR. — Cultivated  on  plain  nutrient  agar,  after  twenty- 
four  to  forty-eight  hours  at  37°  C.,  the  deep  colonies  are  hardly  visible 
to  the  eye.  Under  the  microscope  they  appear  light  yellow  or  brown 
in  color  and  finely  granular.  The  surface  colonies  are  large,  equalling 
in  size  those  of  streptococci,  but  are  usually  more  transparent.  If  blood 
serum  or  ascitic  fluid  be  added  to  the  agar  the  individual  colonies  are 
larger  and  closer  together,  and  the  growth  is  more  distinct  in  conse- 
quence and  of  a  grayish  color.  The  surface  colonies  are  almost  circular 
in  shape  under  a  magnification  of  60  diameters,  finely  granular  in  struc- 
ture, and  may  have  a  somewhat  darker,  more  compact  centre,  surrounded 
by  a  paler  marginal  zone.  With  high  magnification  cocci  in  twos  and 
short  rows  often  distinctly  separated  are  seen  at  the  edges.  In  stick 
cultures  minute  transparent  drops  appear  along  the  line  of  puncture. 

GROWTH  ox  GELATIX. — The  growth  on  gelatin  is  slow,  if  there  is 
any  development  at  all,  owing  to  the  low  temperature — viz.,  24°  to 
27°  C. — above  which  even  the  most  heat-resistant  gelatin  will  melt. 
The  gelatin  is  not  liquefied. 

GROWTH  ox  BLOOD  SERUM. — The  growth  on  Loeffler's  blood-serum 
mixture  is  very  similar  to  that  on  agar,  but  somewhat  more  vigorous 
and  characteristic,  appearing  on  the  surface  as  a  delicate  layer  of  dew- 
like  drops. 

GROWTH  IN  BOUILLON. — In  bouillon,  at  the  end  of  twelve  to  twenty- 
four  hours  in  the  incubator,  a  slight  cloudiness  of  the  liquid  will  be 
found  to  have  been  produced,  due  to  the  development  of  the  micrococci. 
On  microscopic  examination  these  can  be  seen  to  be  arranged  in  pairs 
or  longer  or  shorter  chains.  In  two  or  three  days  the  medium  again 
becomes  transparent  owing  to  the  subsidence  of  the  cocci.  After  one 
or  two  transplantations  the  pneumococci  frequently  fail  to  grow. 

SPECIAL  MEDIA. — Fraenkel  was  the  first  to  draw  attention  to  the 
fact  that  this  organism  soon  loses  its  reproductive  power  when  grown 
on  ordinary  culture  media,  and  more  particularly  solid  media.  In  fluid 
media  the  vitality  is  not  quite  so  quickly  lost;  but  even  here  it  is  found 
advisable  to  transplant  fresh  cultures  every  day.  When  cultures  are 
grown  on  serum-free  media  the  vitality  of  some  cultures  may  indeed 
be  indefinitely  prolonged;  but  after  transplantation  through  several 
generations  it  is  found  that  the  cultures  begin  to  lose  in  virulence, 
and  that  they  finally  become  non-virulent.  In  order  to  restore  this 
virulence,  or  to  keep  it  from  becoming  attenuated,  it  is  necessary  to 
interrupt  the  transplantation  and  pass  the  organism  through  the  bodies 
of  susceptible  animals. 

With  the  view  of  overcoming  these  obstacles  in  the  way  of  culti- 
vating this  rnicrococcus,  several  special  media  have  been  proposed  by 
various  experimenters  in  the  place  of  the  ordinary  culture  media.  The 


352  BACTERIA  PATHOGENIC  TO  MAN 

best  fluid  medium  for  the  growth  of  the  pneumococcus  is  Marmorek's 
mixture,  consisting  of  bouillon  2  parts  and  ascitic  or  pleuritic  fluid  1 
part.  In  this  fluid  pneumococci  grow  well,  and  cultures  when  preserved 
in  a  cool  place  and  prevented  from  drying  retain  their  vitality  and  also 
their  virulence  for  a  number  of  weeks.  Lambert  has  found  cultures 
in  this  medium  alive  and  fully  virulent  after  eight  months. 

Loeffler's  blood-serum  mixture  is  a  good,  solid  tube  medium  for 
making  cultures,  and  is  very  convenient  and  useful  at  autopsies.  One- 
and-one-half  per  cent,  fluid  nutrient  agar  mixed  with  one-third  its 
quantity  of  warm  ascitic  fluid  makes  an  excellent  plate  medium. 

Hiss  SERUM  MEDIA  WITH  AND  WITHOUT  INULIN.— -These  are  very 
useful.  The  inulin  is  fermented  by  typical  pneumococci  with  coagula- 
tion of  the  serum.  While  most  streptococci  fail  to  ferment  the  inulin. 
This  medium  is,  therefore,  of  considerable  diagnostic  value, 

Nutrient  agar,  with  fresh  rabbit  blood  smeared  over  it  makes  an 
excellent  medium  for  growth,  but  prevents  the  development  of  agglu- 
tinable  substance.  On  this  medium,  in  a  moist  atmosphere  at  36°  C., 
the  cultures  retain  their  viability  and  virulence  for  rabbits  for  months,1 

CALCIUM  BROTH  WITH  OR  WITHOUT  DEXTROSE. — This  medium  has 
proven  of  great  value  for  the  propagation  of  cultures  where  agglutina- 
tion tests  are  to  be  carried  out.  The  addition  of  a  small  piece  of 
marble  to  each  tube  of  broth  is  the  most  satisfactory  way  of  preparing 
it.  Marble  broth  for  this  purpose  was  suggested  independently  by 
Bolduan  and  Hiss. 

RESISTANCE  TO  LIGHT,  DRYING,  AND  GERMICIDAL  AGENTS. — On 
artificial  culture  media  the  pneumococci  tend  to  die  rapidly.  This  is 
partially  due  to  the  acid  produced  by  their  growth.  In  sputum  they 
live  much  longer. 

Pneumonic  sputum  attached  in  masses  to  clothes,  when  dried  in  the 
air  and  exposed  to  diffuse  daylight,  retains  its  virulence,  as  shown  by 
injection  in  rabbits,  for  a  period  of  nineteen  to  fifty-five  days.  Exposed 
to  direct  sunlight  the  same  material  retains  its  virulence  after  but  a 
few  hours'  exposure.  This  retention  of  virulence  for  so  long  a  time 
under  these  circumstances  is  accounted  for  by  the  protective  influence 
afforded  by  the  dried  mucoid  material  in  which  the  micrococci  were 
embedded.  Guarnieri  observed  that  the  blood  of  inoculated  animals, 
wrhen  rapidly  dried  in  a  desiccator,  retained  its  virulence  for  months; 
and  Foa  found  that  fresh  rabbit  blood,  after  inoculation  and  cultivation 
in  the  incubator  for  twenty-four  hours,  when  removed  at  once  to  a  cool, 
dark  place,  retained  its  virulence  for  sixty  days.  There  a^re  many  con- 
ditions, therefore,  in  which  the  virulence  of  the  micrococcus  is  retained 
for  a  considerable  length  of  time.  To  germicidal  agents  pneumococci 
are  very  sensitive.  The  fine  spray  expelled  in  coughing  and  loud  speak- 
ing soon  dries  so  completely  that  no  pneumococci  usually  survive  after 
two  hours.  The  same  is  true  for  dust  that  remains  suspended  in  the  air. 

1  The  green  color  produced  by  all  pneumococci  in  blood-agar  media,  and  showing  especially  well 
in  poured  blood-agar  plates  is  not  diagnostic  of  this  organism,  as  some  strains  of  streptococci  produce 
.just  as  intense  a  green. 


THE  DIPLOCOCCUS  OF  PXEUMOMA  353 

Pathogenesis  in  Animals. — Most  strains  of  the  micrococcus  lanceolatus 
are  moderately  pathogenic  for  numerous  animals;  mice  and  rabbits  are 
the  most  susceptible,  indeed  some  strains  are  intensely  virulent  for 
these  animals,  while  guinea-pigs  and  rats  are  much  less  susceptible. 
Pigeons  and  chickens  are  refractory.  In  mice  and  rabbits  the  subcuta- 
neous injection  of  small  or  moderate  quantities  of  pneumonic  sputum  in 
the  early  stages  of  the  disease,  or  of  a  twenty-four  hour  ascitic  broth  cul- 
ture from  such  sputum,  or  of  a  pure,  virulent  ascitic  broth  culture  of  the 
micrococcus,  usually  results  in  the  death  of  these  animals  in  from  twenty- 
four  to  forty-eight  hours.  The  course  of  the  disease  produced  and  the 
post-mortem  appearances  indicate  that  it  is  a  form  of  septicaemia — what 
is  known  as  sputum  septicaemia.  After  injection  there  is  loss  of  appetite 
and  great  debility,  and  the  animal  usually  dies  some  time  during  the 
second  day  after  inoculation.  The  post-mortem  examination  shows  a 
local  reaction,  which  may  be  of  a  serous,  fibrinous,  hemorrhagic,  necrotic, 
or  purulent  character;  or  there  may  be  combinations  of  all  of  these 
conditions.  The  blood  of  inoculated  animals  immediately  after  death 
often  contains  the  micrococci  in  very  large  numbers.  For  microscopic 
examination  they  may  be  obtained  from  the  blood  of  the  veins, 
arteries,  or  cavities  of  the  heart,  and  usually  from  the  pleural  and 
peritoneal  exudations  when  these  are  present. 

True  localized  pneumonia  does  not  usually  result  from  subcutaneous 
injections  into  susceptible  animals,  but  injections  made  through  the 
thoracic  walls  into  the  substance  of  the  lung  may  induce  a  typical 
fibrous  pneumonia.  This  was  first  demonstrated  by  Talamon,  who 
injected  the  fibrinous  exudate  of  croupous  pneumonia,  obtained  after 
death  or  drawn  during  life  from  the  hepatized  portions  of  the  lung, 
into  the  lungs  of  rabbits.  Wadsworth  showed  that  by  injecting  virulent 
pneumococci  into  the  lungs  of  rabbits  which  had  been  immunized,  a 
typical  lobar  pneumonia  was  excited,  the  general  bactericidal  property  of 
the  blood  being  sufficient  to  prevent  the  general  invasion  of  the  bacteria. 

Attenuation  of  Virulence. — This  may  be  produced  in  various  ways. 
The  loss  of  virulence  which  occurs  when  the  micrococcus  is  trans- 
planted through  several  generations  in  culture  fluid  containing  no 
blood  has  already  been  referred  to.  An  apparent  attenuation  of 
virulence  takes  place  also  spontaneously  in  the  course  of  pneumonia. 
It  has  been  shown  by  daily  puncture  of  the  lung  in  different  stages  of 
the  pneumonic  process  that  the  virulence  of  the  organism  diminished 
as  the  disease  progresses,  and  that  the  nearer  the  crisis  was  approached 
the  more  attenuated  it  became.  This  attenuation  is  probably  only 
apparent.  So  many  more  micro-organisms  are  living  in  each  cubic 
centimetre  of  fluid  during  the  early  stages  of  a  pneumonia  that  much 
smaller  quantities  kill.  If  a  little  sputum  be  taken  at  different  periods 
in  the  disease  and  planted  in  ascitic  bouillon  the  resultant  cultures  will 
not  vary  greatly  in  virulence.  The  average  virulence  of  cultures  made 
from  pneumonic  sputum  is  greater  than  in  those  from  normal  sputum. 

Restoration  and  Increase  of  Virulence. — The  simplest  and  perhaps  the 
most  reliable  method  of  restoring  lost  virulence  for  any  susceptible 

23 


354  BACTERIA  PATHOGENIC  TO  MAN 

animal  is  by  passage  through  the  bodies  of  highly  susceptible  animals 
of  the  same  species.  Growth  in  fresh  blood  also  increases  it  for  the 
homologous  animal. 

Toxin  Production. — We  have  little  exact  knowledge  upon  the  nature 
of  the  substances  produced  by  or  through  the  growth  of  the  pneumo- 
cocci  in  animal  tissues  or  artificial  media. 

Occurrence  in  Man  during  Health. — It  is  probable  that  in  crowded 
communities  the  pneumococcus  is  present  on  the  mucous  membranes 
of  most  persons.  We  have  found  it  generally  present  not  only  in  the 
throats  of  persons  living  in  New  York  City,  but  also  in  those  of  persons 
living  on  farms  and  in  the  Adirondack  Mountains.  It  is  commonly 
present  only  on  the  mucous  membranes  of  the  bronchi,  trachea,  pharynx, 
and  nostrils.  The  healthy  lung  seems  to  be  generally  free  from  it. 
Judging  from  animal  tests  it  is  very  possible  that  the  virulence  for  man 
of  the  organisms  present  in  health  is  much  less  than  the  virulence  of 
those  in  a  pneumonic  lung. 

Presence  of  Pneumococci  in  Lobar  and  Bronchopneumonia. — Fully  95 
per  cent,  of  characteristic  cases  of  lobar  pneumonia  are  due  primarily 
to  characteristic  or  atypical  pneumococci.  Usually  no  other  bacteria 
are  obtained  from  the  lungs.  Atypical  cases  are  frequently  due  to 
pneumococci,  but  they  may  be  due  to  streptococci,  influenza  bacilli, 
etc..  The  more  recent  the  infection  the  greater  is  the  number  of  bac- 
teria found  in  the  diseased  lung  area.  As  the  disease  progresses  these 
decrease  in  number  until  finally  at  the  crisis  they  disappear  from  the 
tissues,  though  at  this  time  and  long  after  convalescence  they  may  be 
present  in  the  sputum.  In  atypical  forms  of  pneumonia  they  may 
remain  longer  in  the  tissues,  and  in  walking  pneumonia  they  may  be 
absent  in  the  original  centres  of  infection  or  present  only  as  attenuated 
varieties,  while  the  surrounding,  newly  formed  foci  may  contain  fully 
virulent  cocci.  It  has  been  shown  by  Netter  that  more  than  one-half 
of  the  cases  of  bronchopneumonia,  whether  primary,  or  secondary  to 
some  other  disease,  as  measles  and  diphtheria,  both  in  children  and 
adults,  are  due  to  the  diplococcus  of  pneumonia.  Others,  such  as 
Pearce,  have  found  other  micro-organisms,  especially  the  streptococci, 
in  the  majority  of  cases.  These  findings  will  be  considered  at  the  end 
of  the  chapter. 

The  pneumococci  are  found  partly  in^  the  alveoli  and  bronchioles 
of  the  inflamed  lung  and  partly  in  the  lymph  channels  and  blood  capil- 
laries. Most  of  the  organisms  are  found  free,  but  a  few  are  found  in 
the  leukocytes.  Through  the  lymph  channels  they  find  their  way  to 
the  pleura  and  to  adjacent  lymph  glands.  From  the  capillaries  they 
find  their  way  to  the  general  blood  current,  and  thus  to  distant  parts 
of  the  body.  In  about  20  per  cent,  of  cases  the  pneumococci  are  so 
abundant  that  they  can  be  found  in  cultures  made  from  5  to  10  c.c.  of 
blood.  In  a  number  of  instances  the  foetus  has  been  found  infected. 

Occurrence  in  Inflammations  Complicating  Pneumonia. — In  every  case 
of  lobar  pneumonia  and  in  most  cases  of  bronchopneumonia  pleurisy 
is  developed,  which  is  excited  by  the  same  micro-organism  that  was 


s 


IVERS1TY  J 

' 

THE  DIPl.'n  »<  ,  t   x  ,,/.-   /'YAT.UOA7.1  355 

predominant  in  the  pneumonia.-  With  pneumococci  the  exudate  is 
usually  moderate  and  of  a  fibrinous  character,  but  may  be  more  abun- 
dant and  of  a  serofibrinous  or  purulent  character.  When  the  pleurisy 
is  marked  it  is  more  apt  to  continue  after  the  cessation  of  the  pneu- 
monia. Pleurisy  due  to  pneumococci  is  more  apt  to  go  on  to  sponta- 
neous recovery  than  that  due  to  streptococci  or  staphylococci. 

The  most  frequent  pneumococcic  infections  next  to  pleurisy,  following 
a  pneumonia,  are  those  of  the  pericardium,  endocardium,  and  meninges, 
and  these  not  infrequently  arise  together.  Pneumococcic  inflammations 
of  the  heart  valves  are  apt  to  be  followed  by  extensive  necrosis  and 
growth  of  vegetations.  In  these  cases  pneumococci  can  sometimes 
be  found  in  the  blood  for  many  weeks.  Pericarditis  due  to  pneumo- 
cocci is  a  frequent  complication,  but  is  usually  very  slightly  developed. 
Meningitis  due  to  pneumococci  may  be  either  fibrinous  or  purulent  or 
both.  Arthritis  and  periarthritis  are  rarer  complications  of  a  pneu- 
mococcic pneumonia.  Besides  moderate  parenchymatous  inflammation 
of  the  kidney,  which  occurs  in  most  cases  of  pneumonia,  well-marked 
inflammation  may  occur  in  which  pneumococci  exist  in  the  kidney 
tissues  in  large  numbers.  Osteomyelitis  and  otitis  media  are  not  very 
infrequent. 

How  is  the  pneumococcus  conveyed  from  its  original  seat  in  the  lungs 
to  distant  internal  organs?  Chiefly  by  means  of  the  bloodvessels  and 
lymphatics,  in  both  of  which  it  has  been  found  in  great  numbers.  Proof 
enough  of  its  conveyance  through  the  lymphatics  is  afforded  by  the 
frequent  occurrence  of  inflammations  of  the  serous  membranes  com- 
plicating pneumonia;  but  two  cases  in  particular  have  been  reported 
by  Thue  of  pleurisy  and  pericarditis  following  pneumonia  in  which 
the  lymph  capillaries  have  been  found  to  be  filled  with  diplococci,  as  if 
injected.  Their  presence  in  the  blood  after  death  has  been  amply  proved 
by  numerous  investigations.  In  many  instances  they  have  been  recovered 
from  the  blood  during  life.  Lambert,  as  a  rule,  found  them  in  all  fatal 
cases  twenty-four  to  forty-eight  hours  before  death.  This  examination 
has  considerable  prognostic  value,  as  nearly  all  cases  in  which  the  pneu- 
mococcus is  found  end  fatally.  This  micrococcus  has  been  shown  ex- 
perimentally to  be  capable  of  producing  various  forms  of  septicaemia- 
local  phlegmonous  inflammations,  peritonitis,  pleuritis,  and  meningitis. 
A  further  proof  of  the  transmission  of  this  organism  by  means  of  the 
blood  is  given  by  Foa  and  Bordoni-Uffreduzzi  in  their  investigations 
into  intrauterine  infection  in  pneumonia  and  meningitis.  These  inves- 
tigators have  demonstrated  the  presence  of  the  micrococcus  lanceolatus 
in  fetal  and  placental  blood  and  in  the  uterine  sinuses  in  maternal 
pneumonia.  There  being  no  question,  therefore,  as  to  the  possibility 
of  the  conveyance  of  the  infective  agent  by  means  of  the  blood  and  the 
lymph  to  all  parts  of  the  body,  we  need  not  wonder  at  the  multiplicity 
of  the  affections  complicating  a  pneumonia,  which  are  caused  by  this 
micrococcus;  and  not  only  the  secondary,  but  also  the  primary  diseases, 
as  of  the  brain  and  meninges,  may  be  explained  in  the  same  way. 
Knowing  that  the  saliva  and  nasal  secretions  under  normal  conditions 


356  BACTERIA  PATHOGENIC  TO  MAN 

so  frequently  afford  a  resting  place  for  the  micrococci,  we  have  only 
to  assume  the  production  of  a  suitable  culture  medium  for  these  para- 
sites in  the  body,  brought  about  by  an  abnormal  condition  of  the  mucous 
membranes  from  exposure  to  cold,  or  a  reduction  of  the  vital  resisting 
power  of  the  tissue  cells  in  any  of  the  internal  organs,  caused  by  disease, 
traumatism,  excesses  of  various  kinds,  etc.,  to  comprehend  readily  how 
an  individual  may  become  infected  with  pneumococci,  either  primarily 
affecting  the  lungs  and  secondarily  other  organs  in  the  body,  or  pri- 
marily attacking  the  middle  ear,  the  pericardial  sac,  the  pleura,  the 
serous  cavities  of  the  brain,  etc. 

Presence  in  Inflammatory  Processes  Not  Secondary  to  Pneumonia. — It 
is  now  known  that  the  pneumococcus  may  infect  and  excite  disease  in 
many  tissues  of  the  body  independent  of  any  preliminary  localization 
in  the  lung.  As  a  rule,  these  processes  are  acute  and  usually  run  a 
shorter  and  more  favorable  course  than  similar  inflammations  due  to 
the  streptococci. 

The  most  frequent  primary  lesions  excited  by  the  pneumococcus 
after  lobar  pneumonia,  bronchopneumonia,  and  bronchitis  are  probably 
meningitis,  otitis  media,  endocarditis,  pericarditis,  rhinitis,  tonsillitis, 
conjunctivitis,  and  keratitis;  septicaemia,  arthritis,  and  osteomyelitis; 
inflammations  of  the  epididymis,  testicles,  and  Fallopian  tubes;  peri- 
tonitis, etc. 

Pneumococcic  peritonitis  is  not  so  very  infrequent.  The  exudate 
is  usually  seropurulent. 

Conjunctivitis  due  to  pneumococci  frequently  occurs  in  epidemic 
form  and  is  frequently  associated  with  a  rhinitis. 

From  statistics  collected  by  Netter  the  following  percentages  of 
diseases  were  caused  by  the  pneumccoccus : 

Pneumonia          .         .         .         .         .         .     65.9  per  cent,  in  adults. 

Bronchopneumonia     .....     15.8 

Meningitis  .......     13.0 

Empyema   .         .         .         .         .         .         .8.5 

Otitis  media         ......       2.4 

Endocarditis        .         .         .         .         .         .1.2 

In  46  consecutive  pneumococcus  infections  in  children  there  were: 

Otitis  media 29  cases. 

Bronchopneumonia     .          .         .         .          .         >         .  12      " 

Meningitis 2      " 

Pneumonia          .         .         .         .         .         .         .         .  1      " 

Pleurisy .       1      " 

Pericarditis         .         .         .         .         .         .         .         .         .       1      " 

The  pneumococcus  and  streptococcus  are  the  two  most  frequent 
organisms  found  in  otitis  media.  The  cases  due  to  the  pneumococcus 
are  apt  to  run  the  shorter  course,  but  have  a  tendency  to  spread  to  the 
meninges  and  cause  a  meningitis.  The  pneumococci  may  also  find 
their  way  into  the  blood  current. 

In  bronchitis  the  pneumococcus  is  frequently  met  with  alone  or  in 


TIIK  DIPLOCOCCUS  OF  PXEUMOMA  357 

combination  with  the  streptococcus,  the  influenza  bacillus,  or  other 
bacteria. 

In  certain  epidemics  pneumococcic  bronchitis  and  pneumonia  simu- 
late influenza  very  closely  and  cannot  be  dilYerentiated  except  by  bac- 
teriological examinations. 

Primary  pneumococcic  pleurisy  is  frequent  in  children ;  it  is  very  often 
purulent,  but  may  be  serous  or  serofibrinous.  Its  prognosis  is  better 
than  that  in  cases  due  to  other  organisms.  Frequently  we  have  strep- 
tococci and  staphylococci  associated  with  the  pneumococci. 

Varieties  of  the  Pneumococcus. — As  among  all  other  micro-organisms 
minutely  studied,  different  strains  of  pneumococci  show  quite  a  wide 
range  of  variation  in  morphology  and  virulence.  Some  of  the  variations 
are  so  marked  and  so  constant  that  they  make  it  necessary  to  recognize 
several  distinct  varieties  of  the  pneumococcus,  and  to  class  as  pneumo- 
cocci certain  varieties  which  have  before  this  been  classed  as  strepto- 
cocci— e.  g.,  the  so-called  streptococcus  mucosus  capsulatus  (streptococcus 
mucosus  Schottmiiller),  when  first  isolated  from  pneumonic  exudate  or 
elsewhere,  and  planted  on  artificial  culture  media  containing  serum, 
grows  as  a  rounded  coccus  with  a  small  dense  distinct  capsule,  princi- 
pally in  short  or  medium  chains;  it  produces  a  large  amount  of 
mucus-like  zooglia,  forming  very  large  spreading  colonies;  it  promptly 
coagulates  fluid  serum  media  containing  inulin.  It  is  also  very  virulent 
for  mice,  but  only  moderately  virulent  for  rabbits.  After  a  number  of 
culture  generations  on  ordinary  nutrient  agar  it  apparently  loses  most 
of  these  characteristics.  It  then  grows  in  small  colonies  principally  as 
naked  diplococci  whi?h  may  be  elongated  and  pointed,  produces  no 
zooglia,  and  loses  most  of  its  virulence  for  mice  and  rabbits.  It  still 
coagulates  inulin  serum  media,  and  when  transferred  to  serum  media 
regains  its  former  morphological  characteristics.  For  these  reasons  we 
consider  this  organism  a  distinct  variety  of  the  pneumococcus.  This 
variety  of  pneumococcus  has  been  isolated  by  us  from  the  lungs  after 
death  following  lobar  pneumonia,  out  of  twenty  consecutive  autopsies, 
as  the  only  organism  present  twice,  and  with  another  variety  of  pneu- 
mococcus once.  Together  with  other  varieties  it  was  isolated  from 
four  out  of  twenty  specimens  of  pneumonic  sputum,  and  from  sixty 
specimens  of  normal  throat  secretion  five  times. 

Another  group  of  pneumococci  quite  constantly  produces  large  forms 
and  large  capsules.  Still  another  group  produces  principally  small 
forms  and  small  capsules.  Another  group  might  be  made  of  morpho- 
logically typical  pneumococci  which  do  not  coagulate  inulin  serum 
media. 

Immunity. — Early  in  the  history  of  this  organism  experiments  were 
begun  for  the  production  of  immunity  in  animals  by  means  of  preventive 
inoculations.  Later  it  was  found  that  after  successive  injections  of 
gradually  increasing  doses  of  virulent  pneumococci  into  certain  animals 
(horse,  sheep,  goat,  rabbit),  a  serum  of  some  protective  and  curative 
power  in  experimental  animals  was  obtained.  The  mode  of  action  of 
this  serum  is  still  the  subject  of  study.  According  to  Wright,  Neufeld, 


358  BACTERIA  PATHOGENIC  TO  MAN  - 

and  others,  its  activity  is  due  to  the  presence  of  certain  substances 
called  opsonins  (Wright),  or  bacteriotropic  substances  (Neufeld),  which 
act  on  the  bacteria  in  such  a  way  as  to  prepare  them  for  ingestion  by 
the  phagocytes. 

Agglutination  Reactions. — It  has  been  shown  that  the  specific  serum 
obtained  by  the  above  method  may  contain  a  considerable  quantity  of 
agglutinating  substances  for  the  strain  of  pneumococcus  inoculated  and 
for  certain  other  strains,  but  not  for  all.  In  the  case  of  the  pneumo- 
coccus mucosus  (streptococcus  mucosu-s  Schottmiiller)  we  have  found 
that  all  of  the  strains  tested  by  us  were  agglutinated  in  high  dilutions 
in  the  serum  obtained  after  the  inoculation  of  one  strain. 

Therapeutic  Experiments. — The  number  of  cases  reported  in  which 
the  blood-serum  of  animals  artificially  immunized  against  pneumonic 
infection  has  been  used  for  the  treatment  of  the  disease  in  human 
beings,  although  numerous  has  not  led  to  the  formation  of  a  definite 
opinion  as  to  the  final  value  of  this  as  a  therapeutic  agent.  In  the  cases 
we  have  observed  there  has  been  in  some  a  slight  immediate  lowering 
of  the  temperature;  in  others  no  apparent  change.  As  a  rule,  the  cases 
did  rather  better  than  was  expected,  but  certainly  no  striking  curative 
effects  were  apparent.  The  cases  did  not  develop  pneumococcus  blood 
infection,  and  it  seems  probable  that  the  serum  may  be  able  to  prevent 
a  general  infection  from  taking  place  from  the  diseased  lung,  even 
though  it  may  fail  to  influence  the  local  process.  It  has  also  been  shown 
that  these  injections  of  antipneumococcic  serum  are  practically  harmless. 
In  pneumococcus  septicaemia  no  marked  results  have  been  seen.  The 
majority  who  received  the  injections,  as  well  as  those  not  receiving 
them,  died. 

The  Pneumobacillus  of  Friedlander. 

This  bacillus  discovered  by  Friedlander  (1883)  is  now  known  to  occur 
frequently  as  a  mixed  infection  in  cases  of  phthisis,  fibrinous  pneumonia, 
and  in  rare  instances  as  the  only  exciting  factor  in  pneumonia.  It  is 
also  not  infrequently  found  in  the  mucous  membranes  of  the  mouth 
and  air  passages  of  healthy  individuals. 

Morphology. — Short  bacilli  with  rounded  ends,  often  resembling  micro- 
cocci,  especially  in  recent  cultures;  commonly  united  in  pairs  or  in 
chains  of  four,  and,  under  certain  circumstances,  surrounded  by  a  trans- 
parent capsule.  This  capsule  is  not  seen  in  preparations  made  from 
artificial  culture  media,  but  is  visible  in  well-stained  preparations  from 
the  blood  of  an  inoculated  animal. 

Friedlander's  bacillus  stains  readily  with  the  aniline  colors,  but  is  not 
stained  by  Gram's  method. 

Biology. — An  aerobic,  non-motile,  non-liquefying  bacillus;  also  facul- 
tative anaerobic;  does  not  form  spores.  In  gelatin  stick  cultures  it 
presents  the  "nail-shaped"  growth  first  described  by  Friedlander,  which 
is  not,  however,  peculiar  to  this  bacillus.  Gas  bubbles  occasionally 
develop  in  gelatin,  and  in  old  cultures  the  gelatin  acquires  a  distinct 


THE  PNEUMOBACILLUS  OF  FRIEDLANDER  359 

brownish  coloration.  This  latter  characteristic  distinguishes  the  growth 
of  this  bacillus  from  that  of  the  bacillus  aerogenes,  which  is  otherwise 
very  similar  to  it  morphologically  and  culturally.  On  gelatin  plates 
colonies  appear  at  the  end  of  twenty-four  hours  as  small  white  spheres, 
which  rapidly  increase  in  size.  These  colonies,  when  examined  by  a 
low-power  lens,  present  a  somewhat  irregular  outline  and  a  slightly 
granular  appearance.  The  growth  on  agar  is  in  quite  large  and  moist 
grayish  colonies.  On  blood  scrum  abundant,  grayish-white,  viscid  masses 
arc  developed.  The  growth  on  potato  is  luxuriant — a  thick,  yellowish- 
white,  glistening  layer  rapidly  covering  the  entire  surface.  Milk  is  not 
coagulated.  Indol  is  produced  in  bouillon  or  peptone  solutions.  Milk- 
sugar  and  glucose  are  fermented.  Growth  occurs  at  16°  to  20°  C.,  but 
is  more  rapid  at  37°  C. 

Pathogenesis. — Friedlander's  bacillus  is  pathogenic  for  mice  and 
guinea-pigs,  less  so  for  dogs,  and  rabbits  are  apparently  immune.  On 
autopsy  after  death  due  to  inoculation  into  the  lungs,  the  pleural  cavities 
are  found  to  contain  a  seropurulent  fluid,  the  lungs  are  intensely 
congested,  and  in  places  show7  limited  areas  of  red  hepatization ;  the 
spleen  is  considerably  enlarged,  and  bacilli  are  present  in  the  lungs, 
the  pleuritic  fluid,  and  the  blood. 

Friedlander's  bacillus  has  been  found  in  man,  not  only  in  patients 
suffering  from  croupous  pneumonia  and  other  respiratory  diseases,  but 
also  in  healthy  individuals,  and  in  the  outside  world.  Netter  observed 
it  in  4.5  per  cent,  of  the  cases  examined  by  him  in  the  saliva  of  healthy 
individuals,  and  Pansini  in  cases  of  pulmonary  tuberculosis  in  the 
sputum.  Friedlander  believed  that  the  bacillus  described  by  him  was 
the  specific  cause  of  croupous  pneumonia;  but  in  129  cases  examined  by 
Weichselbaum  this  bacillus  was  found  in  only  9;  of  70  cases  examined 
by  Wolf  only  3  showed  the  presence  of  Friedlander's  pneumobacillus. 
It  is  evident,  therefore,  that  though  this  micro-organism  may  be  con- 
cerned in  the  production  of  certain  forms  of  the  disease,  it  is  not  the 
specific  cause  of  croupous  pneumonia.  The  cases  which  are  due 
primarily  to  the  pneumobacillus  are  distinguished,  according  to  Weichsel- 
baum and  Netter,  by  their  peculiarly  malignant  type  and  by  the  viscidity 
of  the  exudate  produced.  This  bacillus  is  also  probably  concerned, 
primarily  or  secondarily,  under  certain  circumstances,  in  the  production 
of  pleurisy,  abscess  of  the  lungs,  pericarditis,  endocarditis,  otitis  media, 
and  meningitis,  in  all  of  which  diseases  it  has  at  times  been  found  to 
be  present. 


CHAPTEK  XXVI. 

MENINGOCOCCUS  OR  DIPLOCOCCUS  (MICROCOCCUS)  INTRACEL- 

LULARIS  MENINGITIDIS,  AND  THE  RELATION  OF  IT  AND 

OF  OTHER  BACTERIA  TO  MENINGITIS. 

IN  the  description  of  the  diplococcus  of  pneumonia  reference  was  made 
to  this  organism  as  the  most  frequent  cause  of  isolated  cases  of  meningitis, 
especially  when  it  complicated  pneumonia.  In  1887  Weichselbaum 
discovered  another  micrococcus  in  the  exudate  of  cerebrospinal  menin- 
gitis in  six  cases,  two  of  which  were  not  complicated  by  pneumonia.  He 
obtained  it  in  pure  cultures,  studied  its  characteristics,  and  showed  that 
this  organism  was  clearly  distinguishable  from  the  micrococcus  lanceo- 
latus,  and  especially  by  its  usual  presence  in  the  interior  of  pus-cells, 
on  which  account  he  called  it  diplococcus  intracellularis  meningitidis. 
The  frequency  of  the  occurrence  of  this  diplococcus  in  meningitis  and 
its  almost  complete  restriction  to  this  disease  affords  sufficient  evidence 
for  the  assumption  that  it  was  concerned  in  its  production.  In  1895 
Jaeger  and  Scheurer  believed  they  found  it  in  the  nasal  secretions  of 
eighteen  living  persons  suffering  from  this  disease  during  an  epidemic. 

It  seems  very  probable  that  in  most  cases  of  primary  meningitis  it 
is  from  the  mucous  membrane  of  the  nasal  cavities  and  the  sinuses 
opening  out  from  them  that  both  the  diplococcus  of  pneumonia  and 
the  micrococcus  intracellularis  find  their  way  through  the  lymph  chan- 
nels to  the  meninges.  The  former  we  know  to  be  almost  constantly 
present  in  the  nasal  cavities,  and  the  latter  we  have  reason  to  believe 
is  not  infrequently  there.  The  prevalence  of  epidemics  in  winter  and 
spring,  a  time  favorable  to  influenza  and  pneumonia,  also  suggests  the 
respiratory  tract  as  the  source  of  the  infection  and  the  place  where  an 
increase  in  virulence  takes  place.  We  do  not  as  yet  know  why  menin- 
gitis follows  in  some  persons  and  not  in  others  after  infection  of  the 
mucous  membranes. 

The  meningococcus  dies  readily  when  dried,  so  that  we  seldom  inhale 
it  except  in  rooms  occupied  by  those  infected.  Such  persons  and 
things  recently  soiled  by  their  nasal  secretion  are  especially  dangerous. 

Morphology. — This  organism  occurs  as  biscuit-shaped  micrococci, 
usually  united  in  pairs,  but  also  in  groups  of  four  and  in  small  masses; 
sometimes  solitary  and  small  degenerated  forms  are  found.  Cul- 
tures resemble  strongly  those  of  gonococci  (see  Fig1.  111).  In  cultures 
more  than  twenty-four  hours  old  larger  and  smaller  forms  occur  and 
some  which  stain  poorly.  These  are  involution  forms.  In  the  exu- 
dation, like  the  gonococcus,  to  which  it  bears  a  close  resemblance  in 
form  and  arrangement,  it  is  distinguished  by  its  presence,  as  a  rule, 


Tin:  M I:\I.\GOCOCCUS  361 

within  the  polynuclear  leukocytes.  It  never  appears  within  the  nucleus 
and  rarely  within  other  cells  (Fig.  110). 

Staining. — It  ,v/«i'/i.v  with  all  the  ordinary  aniline  colors,  but  best  with 
Loeffler's  methylene  blue.  It  is,  as  a  rule,  readily  decolorized  by  Gram's 
solution.  Some  organisms  in  many  cultures  are  more  resistant  than 
others,  but  none  are  definitely  Gram  positive.  It  is  almost  certain 
that  the  positive  cocci  which  have  been  described  as  meningococci 
are  really  contaminating  organisms. 

Biology.— It  grows  between  25°  and  40°  C.,  best  at  about  37.5°  C.,  and 
its  development  is  usually  scanty  on  the  surface  of  nutrient  agar,  but 
sometimes  a  few  colonies  grow  quite  vigorously.  Now  and  then  cultures 
grow  at  23°  C.  or  slightly  less.  It  grows  scarcely  at  all  in  bouillon, 
and  scantily  in  bouillon  plus  one-third  blood  serum.  It  develops  com- 
paratively well  on  Loeffler's  blood-serum  medium  as  used  for  diphtheria 
cultures,  and  on  blood  serum  or  ascitic-fluid  agar. 


Fro. 110 


Diplococcus  intracellularis  ineiiingitidis.    X  1100  diameters. 

Of  the  usual  sugars  the  meningococcus  ferments  dextrose  only  and 
even  this  not  sufficiently  to  coagulate  the  serum  media.  It  grows  on 
5  per  cent,  glycerin  agar  as  well  as  on  plain  agar. 

When  grown  on  nutrient  agar  or  glycerin  agar,  a  tolerably  good 
growth  develops  at  the  end  of  forty-eight  hours  in  the  incubator.  This 
appears  as  a  flat  layer  of  colonies,  about  one-eighth  of  an  inch  in 
diameter,  grayish-white  in  color,  finely  granular,  rather  viscid,  and  non- 
confluent  unless  very  close  together.  On  Loeffler's  blood  serum  the 
growth  forms  round,  whitish,  shining,  viscid-looking  colonies,  with  smooth 
and  sharply-defined  outlines;  these  may  attain  diameters  of  one-eighth 
to  one-sixteenth  of  an  inch  in  twenty-four  hours.  The  colonies  tend  to 
become  confluent  and  do  not  liquefy  the  serum.  From  the  spinal  fluid 
in  acute  cases,  wliere  the  organisms  are  apt  to  be  more  abundant,  a 
great  many  minute  colonies  may  develop  instead  of  a  few  larger  ones. 
On  agar  plates  the  deep-lying  colonies  are  almost  invisible  to  the 
naked  eye;  somewhat  magnified  they  appear  finely  granular,  with  a 


362  BACTERIA  PATHOGENIC  TO  MAN 

dentated  border.  On  the  surface  they  are  larger,  appearing  as  pale 
disks,  almost  transparent  at  the  edges,  but  more  compact  toward  the 
centres,  which  are  yellowish-gray  in  color.  On  blood  agar  or  serum 
agar  the  growth  is  much  more  luxuriant  than  on  plain  agar  and  larger 
than  the  gonococcus.  Not  infrequently  no  growth  is  obtained  when 
the  cerebrospinal  fluid  containing  the  diplococci  is  placed  on  plain 
agar,  and  in  rare  instances  no  growth  appears  when  serum  agar  is 
used.  Cultivated  in  artificial  media  while  it  often  lives  for  weeks,  it 
may  die  within  four  days,  and  requires,  therefore,  to  be  transplanted 
to  fresh  material  at  short  intervals — at  least  every  two  days. 

Resistance. — It  is  readily  killed  by  heat,  sunlight,  and  drying. 

Pathogenesis. — This  organism  does  not  show  marked  pathogenic  power 
for  adult  animals.  It  is  most  pathogenic  for  mice  and  guinea-pigs,  less 
so  for  rabbits  and  dogs.  Subcutaneous  injections  in  animals  when  large 
cause  death ;  intrapleural  or  intraperitoneal  inoculations  in  mice  and 
guinea-pigs,  when  given  in  large  doses  (y1^  to  J  of  a  blood-serum  cul- 
ture), are  usually  fatal.  Intravenous  injections  in  rabbits  have  caused 
the  death  of  the  animal,  but  no  increase  of  diplococci  in  the  blood  or 
characteristic  pathological  changes  have  been  found  as  a  result  of  the 
injections. 

When  mice  are  inoculated  into  the  pleural  or  peritoneal  cavities  they 
usually  fall  sick  and  die  within  thirty-six  to  forty-eight  hours,  showing 
slight  fibrinopurulent  exudation.  In  the  blood  and  enlarged  spleen 
diplococci  are  found  in  small  numbers  and  mostly  free;  in  the  pleuritic 
exudation  they  are  present  in  considerable  quantities,  less  so  in  the 
peritoneal  fluid,  but  then  occurring  in  the  interior  of  pus  cells. 

Certain  experiments  made  by  Weichselbaum  on  dogs,  though  not 
entirely  successful,  are  interesting  as  showing  the  similarity  of  the  disease 
produced  in  them  artificially  with  meningitis  as  occurring  in  man.  The 
three  dogs,  trephined  and  inoculated  subdurally  with  0.5  to  2  c.c.  of  a 
fresh  culture,  all  died :  No.  1  within  twelve  hours,  No.  2  in  three  days, 
and  No.  3  in  twelve  days.  In  Nos.  1  and  2  there  were  found  hypersemia 
of  the  meninges,  with  inflammatory  softening  of  the  brain  at  the  point 
of  inoculation,  which  on  nearer  inspection  proved  to  be  a  true  encepha- 
litic  process.  In  dog  No.  2,  in  which  the  disease  was  of  longer  duration, 
these  changes,  were  the  most  pronounced.  Numerous  diplococci  were 
observed  in  the  sections  removed,  for  the  most  part  free,  but  some  few 
within  the  pus  cells.  In  dog  No.  3,  in  which  the  disease  lasted  twelve 
days,  a  thick,  reddish,  purulent  liquid  was  found  between  the  dura  mater 
and  the  brain  at  the  point  of  inoculation;  in  the  brain  itself  an  abscess 
had  formed,  about  the  size  of  a  hazel-nut,  filled  with  tough,  yellow  pus, 
while  the  abscess  walls  consisted  of  softened  brain  substance  infiltrated 
with  numerous  hemorrhagic  deposits.  The  ventricles  on  that  side 
contained  a  cloudy,  reddish  fluid,  with  flocks  of  pus;  but  no  diplococci 
could  be  demonstrated  in  the  blood  or  exudations.  In  our  experience 
injection  of  a  recent  culture  into  the  spinal  canal  of  very  young  puppies 
is  regularly  followed  by  the  results  noted  by  Weichselbaum.  Such 
efi'ects  are  not  observed  in  older  dogs. 


////:  M I:\I.\GOCOCCUS 

PRESENCE  IN  THE  NASAL  CAVITY  OF  THE  SICK  AND  TIIK  \Vi  I.L.— In 
1  of  his  6  cases  Weichselbaum  succeeded  in  obtaining  in  pure  culture 
diplococci  from  the  nasal  secretion.  Scheurer,  in  his  18  cases,  found 
the  diplococci  in  the  nasal  secretions  of  all  of  them  during  life.  In  50 
healthy  individuals  examined  they  were  found  in  the  nasal  secretions 
of  only  two,  one  being  a  man  suffering  at  the  time  from  a  severe  cold. 
This  man,  it  is  interesting  to  note,  had  been  engaged  in  disinfecting  a 
room  which  had  previously  been  occupied  by  a  patient  with  cerebro- 
spinal  meningitis.  Lately,  there  has  been  a  tendency  to  throw  doubt 
on  these  findings,  but  from  our  experience  in  the  recent  epidemic  in 
New  York,  one  can  state  that  the  meningococci  are  usually  present  in 
great  numbers  in  the  nose  and  nasopharynx  in  most  cases  of  menin- 
gitis dur'ng  the  first  twelve  days  of  illness.  After  the  fourteenth  day 
they  cannot  usually  be  found.  In  one  case  Goodwin  of  our  laboratory 
obtained  them  on  the  sixty-seventh  day.  She  also  found  them  in  five 
persons  out  of  sixty  tested  who  had  been  in  close  contact  with  the  sick, 
and  in  two  of  fifty  medical  students. 

COMPLICATING  INFECTIONS. — Occasionally  we  find  secondary  to  the 
cerebrospinal  meningitis,  and  due  to  the  micrococcus,  inflammations  of 
nasal  cavities  and  their  accessory  sinuses,  also  catarrhal  inflammations 
of  the  middle  ear,  acute  bronchitis,  and  pneumonia.  The  absolute 
determination  of  the  identity  of  the  micrococcus  found  in  these  con- 
ditions has  not  been  established,  so  that  the  above  complications  can 
only  be  considered  as  probably  due  to  this  organism. 

Except  in  cases  of  meningitis  the  micrococcus  has  been  absolutely 
identified  only  in  cases  of  rhinitis.  Several  observers  believe  they  have 
found  it  in  the  diseases  mentioned  above  as  occasionally  complicating 
meningitis. 

MENINGOCOCCI  IN  THE  BLOOD. — Elser  in  forty  cases  examined 
during  the  early  days  of  the  disease  found  them  in  ten. 

AGGLUTINATION  CHARACTERISTICS. — Meningococci  are  agglutinated 
in  dilutions  of  the  blood  serum  of  animals  immunized  to  any  true 
culture.  As  a  rule  dilutions  higher  than  1  :  40  do  not  give  reactions. 
In  the  second  and  third  weeks  of  disease,  agglutination  in  1  :  10  or 
higher  dilutions  of  serum  may  be  obtained. 

SERUM  TREATMENT. — The  use  of  specific  or  other  sera  has  not 
proven  of  value. 

Bacteriological  Diagnosis. — By  means  of  lumbar  puncture,  fluid  can 
readily  be  obtained  from  the  spinal  canal  without  danger.  The  skin 
must  be  thoroughly  cleansed  and  the  needle  aseptic.  The  fluid  should 
be  placed  in  a  sterile  conical  glass  to  settle.  The  sediment  should  be 
used  to  make  smears  to  examine  (1)  for  pus  cells,  (2)  for  tubercle 
bacilli,  and  (3)  for  other  organisms.  By  Gram's  stain  we  are  able  to 
separate  the  three  Gram-positive  organisms  met  with  in  meningitis 
(pneumococcus,  streptococcus,  and  staphylococcus)  from  the  others. 
Of  importance  also  is  the  point  that  the  micrococcus  intracellularis 
is  usually  inside  the  leukocytes  in  the  form  of  diplococci  of  varying 
size,  of  coffee-bean  shape,  or  of  tetracocci,  while  the  pneumococcus  is 


364  BACTERIA  PATHOGENIC  TO  MAN 

frequently  outside  the  cells  and  is  usually  spherical  or  lancet-shaped 
and  frequently  occurs  in  short  chains.  Sometimes  the  bacteria  are 
present  in  very  small  numbers,  and  then  many  smears  must  be  looked 
through  before  a  probable  diagnosis  can  be  made.  In  all  cases  absolute 
certainty  can  only  be  obtained  through  cultures.  Here  plain  nutrient 
agar,  serum  agar,  and  blood-agar  plates  should  be  made,  and,  if  desired, 
tubes  also.  When  considerable  quantities  are  inoculated  upon  these 
media  and  meningococci  are  present,  as  a  rule,  a  greater  or  less  number 
of  colonies  having  the  characteristics  already  described  will  develop. 
The  value,  clinically,  of  the  examination  is  that  about  40  per  cent,  of 
the  cases  due  to  this  coccus  recover,  while  almost  all  of  those  due  to  the 
pneumococcus  and  streptococcus  die. 

In  many  cases  there  are  very  few  diplococci  present  in  the  spinal 
fluid,  so  that  a  failure  to  find  them  in  a  microscopic  examination 
should  not  be  taken  to  prove  that  the  disease  was  not  due  to  this 
organism.  For  cultures  a  considerable  amount  of  fluid  must  be  used, 
for  we  have  found,  as  described  by  Councilman  and  others,  that 
there  may  be  very  few  living  diplococci  even  in  1  c.c.  of  fluid. 

To  obtain  the  fluid  the  patient  should  lie  on  the  right  side  with  the 
knees  drawn  up  and  the  left  shoulder  depressed.  The  skin  of  the 
patient's  back,  the  hands  of  the  operator,  and  the  large  antitoxin 
syringe  should  be  sterile.  The  needle  should  be  4  cm.  in  length,  with  a 
diameter  of  1  mm.  for  children,  and  longer  for  adults. 

The  puncture  is  generally  made  between  the  third  and  fourth  lumbar 
vertebrae.  The  thumb  of  the  left  hand  is  pressed  between  the  spinous 
processes,  and  the  point  of  the  needle  is  entered  in  the  median  line 
or  a  little  to  the  right  of  it,  and  on  a  level  with  the  thumb-nail,  and 
directed  slightly  upward  and  inward  toward  the  median  line.  At  a 
depth  of  3  or  4  cm.  in  children  and  7  or  8  cm.  in  adults  the  needle 
enters  the  subarachnoid  space,  and  on  withdrawing  the  obturator  the 
fluid  flows  out  in  drops  or  in  a  stream.  If  the  needle  meets  a  bony 
obstruction  withdraw  and  thrust  again  rather  than  make  lateral  move- 
ments. Any  blood  obscures  the  microscopic  examination.  The  fluid 
is  allowed  to  drop  into  absolutely  sterile  test-tubes  or  vials  with  sterile 
stoppers.  From  5  to  15  c.c.  should  be  withdrawn.  No  ill  effects  have 
been  observed  from  the  operations.  On  the  contrary  the  relief  of 
pressure  frequently  produces  beneficial  results. 

Organisms  Exciting  Meningitis. — 1.  The  pneumococcus.  This  diplo- 
coccus  is  one  of  the  most  frequent  exciters  of  meningitis,  not  only  when 
it  is  a  primary  disease,  but  also  when  it  is  secondary  to  a  pneumonia, 
otitis,  etc. 

2.  The    streptococcus    pyogenes   and    the   siaphylococcus.    pyogenes. 
Meningitis  due    to  these    organisms  is  almost    always    secondary  to 
some  other  infection,  such  as  otitis,  tonsillitis,  erysipelas,  endocarditis, 
suppurating  wounds  of  scalp  and  skull,  etc. 

3.  The  bacillus  influenzT.     Numerous  doubtful  reports  have  been 
published  of  the  presence  of  influenza  bacilli  in  the  meningeal  exudate. 
Those  that  are  reliable  state  in  almost  every  instance  that  the  menin- 


THE  MICROCOCCUS  CATARRH ALIS  365 

gitis  is  secondary  to  infection  of  the  lungs,  bronchi,  the  nasal  cavities 
or  their  accessary  sinuses. 

4.  The  colon  bacillus,  the  typhoid  bacillus,  that  of  bubonic  plague  and 
of  glanders,  all  may  cause  a  complicating  purulent  meningitis. 

5.  In  isolated  cases  of  meningitis  complicating  otitis  media  and  other 
infections,  other  bacteria,  such  as  the  micrococcus  tetragenus,  the  bacillus 
pyot'i/uncux,  etc.,  may  be  found 

Micrococcus  Catarrhalis  (R.  Pfeiffer). 

Micrococci  somewhat  resembling  meningococci  are  found  in  the 
mucous  membranes  of  the  respiratory  tract.  They  are  believed  at 
times  to  excite  catarrhal  inflammation  of  the  mucous  membranes. 
These  are  at  present  included  under  the  designation  of  micrococcus 
catarrhalis. 

Microscopic  Appearance. — They  usually  occur  in  pairs,  sometimes  in 
fours;  never  in  chains.  The  cocci  are  coffee-bean  in  shape  and  slightly 
larger  than  the  gonococcus,  and  are  negative  to  Gram's  stain. 

The  micrococci  are  not  motile  and  produce  no  spores. 

Cultivation.— They  grow  between  20°  and  40°  C.,  best  at  37°  C.  and 
less  rapidly  at  somewhat  lower  temperatures.  They  develop  on  ordin- 
ary nutrient  agar  as  grayish-white  or  yellowish-white,  circular  colonies  of 
the  size  of  meningococci.  The  borders  of  the  colonies  are  irregular  and 
abrupt  as  though  gouged  out.  They  have  a  mortar-like  consistency. 
On  serum-agar  media  the  growth  is  more  luxuriant.  Gelatin  is  not 
liquefied.  Bouillon  is  clouded,  often  with  the  development  of  a  pellicle. 
Milk  is  not  coagulated,  but  dextrose  serum  media  may  be.  Gas  is  not 
produced. 

Location  of  Organisms. — In  the  secretion  of  normal  mucous  mem- 
branes they  are  occasionally  present.  In  certain  diseased  conditions  of 
the  mucous  membranes  they  may  be  abundant. 

Pathogenic  Effects  in  Animals. — For  white  mice,  guinea-pigs,  and 
rabbits,  some  cultures  are  as  pathogenic  as  meningococci,  while  others 
are  less  so. 

Differential  Points  Separating  them  from  the  Meningococci. — These 
organisms  have  undoubtedly  been  at  times  confused.  Some  assert  that 
the  meningococci  grow  only  above  25°  C.  Many  cord  cultures  of 
meningococci  grow  below  this  point.  Some  assert  that  the  meningo- 
cocci will  not  grow  on  5  per  cent,  glycerin  agar.  Many  undoubted 
cultures  do.  The  probability  is  that  the  organisms  described  by  dif- 
ferent writers  as  micrococcus  catarrhalis  were  not  all  the  same  variety, 
and  some  of  them  were  meningococci. 


CHAPTER  XXVII. 

THE  GONOCOCCUS  OR  MICROCOCCUS  GONORRHOEA— THE 
DUCREY  BACILLUS  OF  SOFT  CHANCRE. 

THE  period  at  which  gonorrhoea  began  to  afflict  man  is  unknown. 
The  earliest  records  make  mention  of  it.  Except  for  a  period  after  the 
fifteenth  century  it  was  generally  recognized  as  a  communicable  dis- 
ease and  laws  were  made  to  control  its  spread.  The  differentiation 
between  the  lighter  forms  of  gonorrhoea  and  some  other  inflammations 
of  the  mucous  membranes  was,  however,  almost  impossible  until  the 
discovery  of  the  specific  micro-organism  by  Neisser,  in  1879. 

The  organism  was  first  observed  in  gonorrhceal  discharges,  and  de- 
scribed by  him  under  the  name  of  "gonococcus ;"  but  though  several 
attempted  to  discover  a  medium  upon  which  it  might  be  cultivated, 
it  was  reserved  for  Bumrn,  in  1885,  to  obtain  it  in  pure  culture  upon 
coagulated  human-blood  serum,  and  then  after  cultivating  it  for  many 
generations  to  prove  its  infective  virulence  by  inoculation  into  man. 
The  researches  of  Neisser  and  Bumm  established  beyond  doubt  that 
this  organism  is  the  specific  cause  of  gonorrhoea  in  man. 

Microscopic  Appearance. — Micrococci,  occurring  mostly  in  the  form 
of  diplococci.  The  bodies  of  the  diplococci  are  elongated,  and,  as 
shown  in  stained  preparations,  have  an  unstained  division  or  inter- 
space between  two  flattened  surfaces  facing  one  another,  which  give 
them  their  characteristic  "  coffee-bean"  or  "  kidney"  shape.  The 
older  cocci  lengthen,  then  become  constricted  in  their  middle  portion, 
and  finally  divide,  making  new  pairs  (Fig.  111).  The  diameter  of  an 
associated  pair  of  cells  varies  according  to  their  stage  of  development 
from  0.8,«  to  l.ftfjt  in  the  long  diameter — average  about  1.25," — by  0.6^ 
to  0.8/>-  in  the  cross  diameter. 

Intracellular  Position  of  Gonococci. — In  gonorrhoea,  during  the  earliest 
stages  before  the  discharge  becomes  purulent,  the  gonococci  are  found 
mostly  free  in  the  serum  or  plastered  upon  the  epithelium  cells, 
but  later  almost  entirely  in  small,  irregular  groups  in  or  upon  the 
pus  cells,  and  always  extranuclear.  With  the  disappearance  of  the 
pus  formation  more  free  gonococci  appear.  Discharge  expressed 
from  the  urethra  usually  contains  more  free  organisms  than  the  natural 
flow.  Gonococci  are  sometimes  irregular  in  shape  or  granular  in 
appearance,  involution  forms,  found  particularly  in  older  cultures  and 
in  chronic  urethritis  of  long  standing.  Single  pus  cells  sometimes  con- 
tain as  many  as  one  hundred  gonococci  and  seem  to  be  almost  bursting 
and  yet  show  but  slight  signs  of  injury.  There  is  still  discussion  as  to 
whether  the  gonococci  actively  invade  the  pus  cells  or  only  are  taken 


THE  GOXOCOCCUS  OR  MICROCOCCUS  GONORRHCE& 


367 


up  by  them.     There  is  no  evidence  that  the  gonococci  are  destroyed 
by  tlie  pus  cells  (Fig.  112). 

Staining.  —  The  gonococcus  stains  readily  with  the  basic  aniline 
colors.  Loeffler's  solution  of  methylene  blue  is  one  of  the  best  staining 
agents  for  demonstrating  its  presence  in  pus,  for,  while  staining  the  gono- 
cocci deeply,  it  leaves  the  cell  protoplasm  but  faintly  stained.  Fuch- 
sin  is  apt  to  overstain  the  cell  substance.  Beautiful  double-stained 
preparations  may  be  made  from  gonorrhceal  pus  by  treating  cover- 
glass  smears  with  methylene  blue  and  eosin.  Numerous  methods  for 
double  staining  have  been  employed,  with  the  object  of  making  a  few 
gonococci  more  conspicuous.  None  of  them  have  any  specific  char- 
acteristics such  as  the  Gram  stain.  It  is  now  established  that  gono- 
cocci from  fresh  cultures  and  from  recent  gonorrhceal  infections  are, 
when  properly  treated  by  Gram's  method,  quickly  and  surely  robbed 
of  their  color.  The  removal  of  the  stain  from  gonococci  in  old  flakes 


FIG.  Ill 


FIG.  112 


Smear  from  pure  culture  of  gonococcus  on  agar. 
X  1100  diameters.    (Heiman.) 


Gonococcus  in  pus  cells. 
X  1100  diameters. 


and  threads  from  chronic  cases  is  not  so  certain.  This  difference  is 
mostly  due  to  the  fact  that  equally  uniform  specimens  cannot  be  pre- 
pared. The  decolorized  gonococci  are  stained  by  dipping  the  films 
for  a  few  seconds  into  a  1  :  10  dilution  of  carbol-fuchsin,  or  a  solu- 
tion of  bismarck  brown  This  staining  should  be  for  as  short  a  time 
as  suffices  to  stain  the  decolorized  organisms.  This  method  of  stain- 
ing cannot  be  depended  upon  alone  to  absolutely  distinguish  the 
gonococcus  from  all  other  diplococci  found  in  the  urethra  and  vulvo- 
vaginal  tract,  for,  especially  in  the  female,  other  diplococci  are  occa- 
sionally found  which  are  also  not  stained  by  Gram's  method.  It 
serves,  howrever,  to  distinguish  this  micrococcus  from  the  common 
pyogenic  cocci,  which  retain  their  color  when  treated  in  the  same  way, 
and  in  the  male  urethra  it  is  practically  certain,  as  no  organism  has 
been  found  in  that  location  which  in  morphology  and  staining  is  iden- 
tical with  the  gonococcus.  It  is  certainly  the  most  distinctive  charac- 
teristic of  the  staining  properties  of  the  gonococcus,  and  it  is  a  test 


368  BACTERIA  PATHOGENIC  TO  MAN 

that  should  never  be  neglected  in  differentiating  this  organism  from 
others  which  are  morphologically  similar. 

Biology. — Grows  best  at  blood  temperature;  the  limits  being  roughly 
25°  and  40°  C.  It  is  a  facultative  anaerobe.  It  is  not  motile  and  produces 
no  spores. 

Culture  Media. — The  gonococcus  requires  for  its  best  growth  the  ad- 
dition to  nutrient  agar  of  a  small  percentage  of  blood  serum  or  some 
equivalent.  The  following  media  have  proven  of  value : 

1.  Human   blood   from   the   sterilized   finger  streaked   on   common 
nutrient  agar. 

2.  Human-blood  serum,   1  part  added  to  and  mixed  with  2  parts 
melted  5  per  cent,  glycerin  nutrient,  1.5  per  cent,  agar  having  a  tem- 
perature of  55°  to  60°  C.    The  whole  after  mixing  being  poured  into 
a  Petri  dish  or  cooled  slanted  in  a  tube.     The  same  proportions  of 
nutrient  broth  and  serum  make  a  suitable  fluid  media. 

3.  Human  ascitic,  pleuritic  or  cystic  fluid  in  same  proportions  as  blood 
serum.     One  per  cent,  glucose  may  be  added. 

4.  Swine  serum  nutrose  media.     Wasserman  strongly  recommends 
this  mixture.    (See  under  Media.)    In  our  hands  it  has  given  good  results. 

5.  Nutrient  or  5  per  cent,  glycerin  agar.     When  considerable  pus  is 
streaked  on  simple  agar  media  a  good  growth  of  gonococci  is  usually 
obtained.     After  continued  cultivation   gonococci   cultures    frequently 
grow  on  media  containing  no  serum.     Some  strains  grow  on  ordinary 
glycerin  or  glucose  nutrient  agar  from  the  start. 

Viability. — Cultures  frequently  die  in  forty-eight  to  seventy-two 
hours  when  kept  at  room  temperature.  In  the  ice-box  they  may  live 
for  several  weeks.  Most  cultures  require  the  serum  media  for  the 
later  cultures, 

Appearance  of  Colonies. — A  delicate  growth  is  characteristic.  At  the 
end  of  twenty-four  hours  there  will  have  developed  translucent,  very 
finely  granular  colonies,  with  scalloped  margin.  The  margin  is  some- 
times scarcely  to  be  differentiated  from  the  culture  medium.  In  color 
they  are  grayish- white,  with  a  tinge  of  yellow.  The  texture  is  finely 
granular  at  the  periphery,  presenting  punctated  spots  of  higher  refrac- 
tion in  and  around  the  centre  of  yellowish  color  (Fig.  113). 

SURFACE  STREAK  CULTURE. — Translucent  grayish-white  growth, 
with  rather  thick  edges. 

Resistance. — The  gonococcus  has  but  little  resistant  power  against 
outside  influences.  It  is  killed  by  weak  disinfecting  solutions  and  by 
desiccation  in  thin  layers.  In  comparatively  thick  layers,  however,  as 
when  gonorrhoeal  pus  is  smeared  on  linen,  it  has  lived  for  forty-nine 
days,  and  dried  on  glass  for  twenty-nine  days  (Heiman).  It  is  killed 
at  a  temperature  over  42°  C. 

Occurrence  of  Gonococci. — Outside  of  the  human  body  or  material 
carried  from  it  gonococci  have  not  been  found. 

Pathogenesis. — Non-transmissible  to  all  animals.  Both  the  living 
and  dead  gonococci  contain  toxic  substances  which  cause  death  or 
injury  when  injected  in  large  quantities. 


THE  GOXOCOCCUS  OR  MICROCOCCUS  GONORRHCE& 

Though  animal  inoculations  are  thus  followed  by  negative  results, 
the  etiological  relation  of  the  gonococcus  to  human  gonorrhoea  has 
been  demonstrated  beyond  question  by  the  infection  of  a  number  of 
healthy  men  with  the  disease  by  the  inoculation  of  pure  cultures  of 
the  micro-organism. 

TOXINS. — In  the  gonococcus  cells  substances  are  present  which  are 
toxic  after  heating  and  contact  with  alcohol.  Injected  in  considerable 
amounts  into  rabbits  they  cause  infiltration  and  often  necrosis.  Applied 
to  the  urethral  mucous  membrane  there  is  produced  an  inflamma- 
tion of  short  duration.  In  gonorrhoea  the  secretion  is  believed  to  be 
due  to  these  intracellular  toxins.  Repeated  injections  give  no  appre- 
ciable immunity.  The  filtrate  of  recent  gonococcus  cultures  contains 
little  or  no  appreciable  toxin.  The  typical  incubation  and  symptoms 
of  the  disease  resulted  in  all  cases  in  the  subjects  experimented  on. 


FIG.  113 


Colonies  ot  gonococci  on  pleuritic  fluid  agar.    (Heiman.) 

Disease  Conditions  Excited  by  Gonococci. — Affections  due  to  this 
organism  are  usually  restricted  to  the  mucous  membranes  of  the 
urethra,  prostate,  neck  of  bladder,  cervix  uteri,  vagina,  and  conjunctiva. 
The  conjunctival,  vaginal,  and  rectal  mucous  membranes  are  much 
more  sensitive  in  early  childhood  than  in  later  life.  Besides  these  tissues 
it  has  been  proven  that  many  others  may  be  infected,  so  that  we  now 
know  that  to  the  gonococci  are  due  many  cases  of  endometritis,  metritis, 
salpingitis,  oophoritis,  peritonitis,  proctitis,  cystitis,  epididymitis,  and 
arthritis.  Abscesses  of  considerable  size,  periostitis,  and  otitis  are  occa- 
sionally due  to  the  gonococcus. 

Endocarditis  and  Septicaemia. — Cases  of  gonococcus  endocarditis  and 
septicaemia  are  not  infrequent.  Gonococcus  septicaemia  may  occur  in 
connection  with  other  localizations  or  alone.  Nearly  every  year  one 
or  two  of  these  cases  is  met  with  in  every  general  hospital.  In  a 
considerable  number  of  cases  where  gonococci  are  obtained  from  the 
blood  the  patients  recover.  The  fever  is  sometimes  typhoid-like  in 
character. 

24 


370  BACTERIA  PATHOGENIC  TO  MAN 

Pavement  epithelium  is  more  difficult  to  infect  than  cylindrical. 
The  gonococcus  gradually  penetrates  the  epithelial  layer  and  produces 
inflammation  of  the  connective  tissue. 

Immunity. — Immunity  in  man  after  recovery  from  infection  seems  to 
be  only  slight  in  amount  and  for  a  short  period  if  present  at  all.  It  is 
known  that  the  urethra  in  man  or  cervix  uteri  in  woman  may  contain 
gonococci  which  lie  dormant  and  may  be  innocuous  in  that  person  for 
years,  but  which  may  at  any  time  excite  an  acute  gonorrhoea  in  another 
individual  or,  under  stimulating  conditions,  in  the  one  carrying  the 
infection. 

Duration  of  Infections  and  of  Contagious  Period. — There  is  no  limit 
to  the  time  during  which  a  man  or  woman  may  remain  infected  with 
gonococci  and  infect  others.  We  have  had  one  case  under  observation 
where  twenty  years  had  elapsed  since  exposure  to  infection,  and  yet  the 
gonococci  were  still  abundant.  It  is  now  well  established  that  most  of 
the  inflammations  of  the  female  genital  tract  are  due  to  gonococci,  and 
the  majority  of  such  infections  are  produced  in  innocent  women  by 
their  husbands  who  are  suffering  from  latent  gonorrhoea. 

Bacteriological  Diagnosis  of  Gonorrhoea. — In  view  of  the  fact  that 
several  non-gonorrhoeal  forms  of  urethritis  exist,  and  also  that  micro- 
cocci  morphologically  similar  to  the  gonococcus  Neisser  are  often 
found  in  the  normal  vulvovaginal  tract,  it  becomes  a  matter  of  great 
importance  to  be  able  to  detect  gonococci  when  present,  and  to  differ- 
entiate these  from  the  non-specific  organisms.  Besides  this,  the  gono- 
cocci which  occur  in  old  cultures  and  in  chronic  urethritis  of  long 
standing  sometimes  take  on  a  very  diversified  appearance.  From  a 
medicolegal  and  social  standpoint,  therefore,  the  differential  diagnosis 
of  the  gonococcus  has  in  certain  cases  a  very  practical  significance. 

There  are  two  methods  of  differential  diagnosis  now  available — 
the  microscopic  and  the  cultural.  Animal  inoculations  are  of  no 
value,  as  animals  are  not  susceptible,  and,  of  course,  human  inocula- 
tions are  generally  impossible.  In  the  microscopic  diagnosis  it  should 
be  borne  in  mind  that  after  the  acute  serous  stage  has  passed,  the  spe- 
cific gonococci  in  carefully  made  preparations  are  always  found  largely 
within  the  pus  cells.  Diplococci  morphologically  similar  to  gonococci 
occurring  in  other  portions  of  the  field  and  outside  of  the  pus  cells 
should  not  be  considered  specific  by  this  test  only.  It  should  also  be 
remembered  that  the  gonococci  are  decolorized  by  Gram's  method, 
while  other  similar  micrococci  which  occur  in  the  urethra  are,  as  a  rule, 
at  least  not  so  decolorized.  Organisms  having  these  characteristics 
can  for  all  practical  purposes  be  considered  as  certainly  gonococci 
if  obtained  from  the  urethra.  From  the  vulvovaginal  tract  the  cer- 
tainty is  not  so  great,  since  other  diplococci  are  occasionally  found  in 
gonorrhceal  pus  from  the  vulvovaginal  tract,  and  very  rarely,  also, 
from  the  urethra,  which  do  not  stain  by  this  method;  here  cultures 
should  also  be  made.  Cover-glass  preparations  from  subacute  or 
chronic  cases  should  be  examined,  if  possible,  with  a  microscope  pro- 
vided with  a  mechanical  stage,  and  films  should  always  be  stained  by 


Y7//:  GO\UCOCCUS  OR  MICROCOCCUS  GOXu/tKIHEJB  371 

both  Loeffler's  methylene-blue  solution  and  by  (Aram's  method,  and 
the  examination  repeated  on  three  consecutive  days.  Should  these 
specimens  prove  negative,  to  exclude  any  possible  doubt  in  the  matter, 
cultures  should  then  be  made  on  human  ascitic  fluid  or  serum  agar, 
poured  in  dishes;  also,  if  with  negative  results,  on  three  consecutive 
days.  Heiman,  who  has  paid  much  attention  to  gonococcus  examina- 
tions, obtains  his  material  by  the  following  method:  in  chronic  urethritis 
he  allows  the  patient  to  void  his  urine  either  immediately  into  two 
sterilized  centrifugal  tubes  or  first  into  two  sterile  bottles.  The  first 
tul>e  will  contain  threads  of  the  anterior  urethra;  the  second  tube  will 
be  likely  to  contain  secretion  from  the  posterior  urethra  and  from  the 
prostate  gland  if,  while  urinating,  the  patient's  prostate  l>e  pressed 
upon  with  the  finger.  Tubes  containing  such  urine  are  placed  in  the 
centrifuge  and  whirled  for  three  minutes  at  twelve  hundred  or  more 
revolutions  per  minute;  the  threads  are  thrown  down.  The  centrifuged 
sediment  will  be  found  to  contain  most  of  the  bacteria  present,  epithelial 
cells,  and,  at  times,  spermatozoa.  Normal  urine  on  being  centrifuged 
at  this  velocity  will  be  found  at  times  to  show  a  slight  turbidity  at  the 
bottom  of  the  tube.  This  will  be  found,  on  microscopic  examination, 
to  consist  of  epithelial  cells,  a  few  leukocytes,  and  some  bacteria. 

The  careful  examination  of  gonorrhceal  threads  stained  by  Gram's 
method  is  a  very  tedious  affair,  as  in  every  instance  no  less  than  three 
cover-glass  preparations  should  be  looked  over  before  the  absence  of 
the  gonococcus  is  considered  probable.  It  would  require  many  hours 
upon  each  and  every  specimen,  especially  if  the  gonococci  are  present 
in  very  small  number,  before  a  reliable  and  conscientious  opinion 
could  be  rendered.  If,  after  all,  a  negative  opinion  is  ventured,  we 
still  are  under  the  necessity  of  proving  that  because  the  threads  which 
we  fished  out  for  the  cover-glass  examination  were  free  from  gonococci 
the  remaining  ones  were  also.  For  this  reason  the  culture  medium 
is  more  sensitive  for  bacteria  than  is  the  cover-glass,  for  we  are  able 
to  plant  each  and  every  thread  of  the  sediment  in  the  centrifugal  tube. 
Results  on  culture  media  are  only  reliable  when  obtained  by  thor- 
oughly trained  bacteriologists  with  suitable  media  and  methods.  Fiir- 
bringer,  in  his  work,  mentions  the  fact  that  in  certain  cases  the  absence 
of  the  gonococcus  in  many  examinations  of  cover-glass  preparations 
is  not  a  positive  proof  that 'the  gonococcus  is  not  present.  The  culture 
methods,  of  course,  presuppose  that  one  has  the  facilities  and  knowl- 
edge to  carry  them  out  successfully,  otherwise  the  microscopic  methods 
are  to  be  used  alone. 

In  acute  cases  where  the  pus  is  abundant  the  specimen  for  examina- 
tion may  be  collected,  when  the  patient  is  before  one,  by  passing  a 
sterilized  platinum-wire  loop  as  far  up  into  the  urethra  as  possible  and 
withdrawing  some  of  the  secretion. 

Occurrence  in  Cultures  from  Chronic  Urethritis. — Heiman  in  61  cases 
found  the  gonococcus  in  14  by  cultures  and  in  !•'>  by  smears.  The 
following  results  were  obtained  by  other  observers  by  cover-glass  prep- 
arations: Goll,  according  to  his  elaborate  article,  examined  1046  cases 


372  BACTERIA  PATHOGENIC  TO  MAN 

of  chronic  urethritis  varying  in  duration  between  four  weeks  to  six  years 
or  more,  finding  gonococci  in  178  cases,  the  remainder  giving  negative 
results.  Neisser,  out  of  143  cases,  varying  in  duration  between  two 
months  and  eight  years,  found  gonococci  in  80  cases. 


BACTERIA  RESEMBLING  GONOCOCCI. 

Bumm  described  a  number  of  micrococci  which  resembled  gonococci 
in  form  and  staining.  These  assume  importance  largely  because 
they  may  be  confused  with  the  gonococcus.  They  occur  on  the  con- 
junctival  and  vaginal  mucous  membranes  and  cause  confusion.  One 
of  these  micro-organisms,  the  micrococcus  catarrhalis  (see  p.  365),  has 
an  importance  of  its  own. 

The  Micrococcus  Melitensis. 

This  micro-organisrn  was  first  discovered  in  a  case  of  Malta  fever  by 
Bruce  in  Malta  in  1887.  The  disease  is  mostly  confined  to  the  shores 
of  the  Mediterranean,  but  cases  of  it  have  been  observed  in  Porto  Rico 
and  the  Philippines.  The  disease  does  not  seem  to  be  directly  trans- 
mitted from  person  to  person. 

Morphology. — Small  rounded  or  slightly  oval  organism  about  15/*  in 
diameter.  It  is  usually  single  or  in  pairs,  but  in  cultures  short  chains 
are  also  met  with.  Durham  has  shown  that  in  old  cultures  bacillary 
forms  occur.  Gorham  believes  he  has  demonstrated  that  the  coccus 
has  one  to  four  flagella. 

Staining. — It  stains  readily  with  the  aniline  dyes  and  is  negative  to 
Gram. 

Cultivation. — At  37°  C.  it  grows  on  nutrient  agar  and  in  broth.  The 
colonies  are  not  usually  visible  until  the  third  day.  They  appear  as 
small  round  disks,  slightly  raised,  with  a  yellowish  tint  in  the  centre. 

Methods  of  Diagnosis. — During  life  the  best  means  of  diagnosis  is  by 
the  agglutination  test.  The  serum  from  different  persons  agglutinates 
in  dilutions  of  from  1 : 10  to  1 : 1000.  Cultures  are  readily  obtained 
from  the  spleen. 

The  Ducrey  Bacillus  of  Soft  Chancre. 

This  bacillus  was  first  specifically  described  and  obtained  :n  pure 
culture  by  Ducrey  in  1889. 

Morphology. — About  1.5,u  long  and  0.4^  thick,  growing  often  in 
chains  and  in  cultures,  sometinies  twisted  together  in  dense  masses. 

It  stains  best  with  carbol-fuchsin,  and  shows  polar  staining. 

Cultural  Characteristics. — The  following  method  of  cultivation  has 
given  the  best  results.  Two  parts  agar  are  liquefied  at  50°  C.  and 
mixed  with  one  part  human,  dog,  or  rabbit  blood.  The  blood  from 
the  cut  carotid  of  a  rabbit  may  be  allowed  to  run  directly  into  the  agar 


THE  DUCREY  BACILLUS  OF  SOFT  CHANCRE  373 

tube,  to  which  the  pus  from  the  ulcerated  bubo  is  then  added  in  proper 
proportion,  and  the  whole  placed  in  the  incubator  at  35°  C.  The  pus 
may  be  obtained  by  puncture  and  aspiration  from  the  unbroken  ulcer, 
or  if  the  ulcer  is  already  open  it  is  first  painted  with  tincture  of  iodine 
and  covered  with  collodion  or  sterile  gauze.  After  twenty-four  to 
forty-eight  hours,  some  pus  having  collected  under  the  bandage,  inocu- 
lations are  made  from  it.  The  bacillus  grows  well  also  in  uncoagulated 
rabbit-blood  s  rum  or  in  condensation  water  of  blood  agar.  In  twenty- 
four  to  forty-eight  hours,  on  the  surface  of  the  media,  well-developed, 
shining,  grayish  colonies,  about  1  mm.  in  diameter,  may  be  observed. 
The  colonies  remain  separate,  but  only  become  numerous  after  further 
transplantation.  The  best  results  are  obtained  when  the  pus  is  taken 
close  to  the  walls  of  the  abscess. 

Glass  smears  show  isolated  bacilli  or  short  parallel  chains  with  dis- 
tinct polar  staining. 

After  the  eleventh  generation  of  the  culture,  and  from  all  old  cul- 
tures, on  inoculation  the  characteristic  soft  chancre  is  produced  in 
man.  According  to  some  observers  animals  cannot  be  infected;  others 
claim  to  have  obtained  positive  results  with  monkeys  and  cats. 

The  organisms  are  especially  characteristic  in  the  water  of  condensa- 
tion from  blood  agar,  the  bacilli  being  thinner  and  shorter,  with  rounded 
ends;  sometimes  long,  wavy  chains  are  found.  In  rabbit-blood  serum 
at  37°  C.  a  slight  clouding  of  the  medium  is  produced  and  small  flakes 
are  formed,  consisting  of  short  bacilli  or  moderately  long,  curved  chains, 
showing  polar  staining. 

The  bacillus  lives  several  weeks  on  blood  agar  at  37°  C.,  but  it 
soon  dies  in  cultures  on  coagulated  serum.  All  other  ordinary  culture 
media  so  far  tried  have  given  negative  results,  and  even  with  the  media 
described  development  is  difficult  and  often  fails  entirely. 

The  chancre  bacillus  possesses  but  little  resistance  to  deleterious  out- 
side influences.  Hence,  the  various  antiseptic  bandages,  etc.,  used  in 
treatment  of  the  affection  soon  bring  about  recovery  by  preventing 
the  spread  of  inoculation  chancre. 


CHAPTER   XXVIII. 

BACILLUS    PYOCYANEUS    (BACILLUS    OF    GREEN    AND    OF    BLUE 

PUS)— BACILLUS  PROTEUS  VULGARIS— GROUP  OF  MALIGNANT 

(EDEMA  BACILLI— BACILLUS  AEROGENES  CAPSULATUS. 

Bacillus  Pyocyaneus. 

THE  blue  and  green  coloration  which  is  occasionally  found  to  accom- 
pany the  purulent  discharges  from  open  wounds  is  usually  due  to  he 
action  of  the  bacillus  pyocyaneus.  According  to  recent  investigations 
this  bacillus  appears  to  be  very  widely  distributed.  It  was  first  obtained 
in  pure  culture  by  Gessard. 

Morphology. — Slender  rods  from  0.3/*  to  I/JL  broad  and  from  2ju  to  6,« 
long;  frequently  united  in  pairs  or  in  chains  of  four  to  six  elements; 
occasionally  growing  out  into  long  filaments  and  twisted  spirals.  The 

FIG.  114 


>•    >•  '  •*** 


Bacillus  pyocyaneus.    (From  Kolle  and  Wassermann.) 

bacillus  is  actively  motile,  a  single  flagellum  being  attached  to  one 
end.  Does  not  form  spores.  Stains  with  the  ordinary  aniline  colors  ; 
does  not  stain  with  Gram's  solution. 

Biology.  —  An  aerobic,  liquefying,  motile  bacillus.  Capable  also  of 
an  anaerobic  existence,  but  then  produces  no  pigment.  Grows  readily 
on  all  artificial  culture  media  at  the  room  temperature,  though  best 
at  37°  C.,  and  gives  to  some  of  them  a  bright-green  color  in  the  pres- 
ence of  oxygen.  In  gelatin-plate  cultures  the  colonies  are  rapidly  de- 
veloped, imparting  to  the  medium  a  fluorescent  green  color;  liquefac- 
tion begins  at  the  end  of  two  or  three  days,  and  by  the  fifth  day  the 


BACILLUS  PYOCYAM-US  375 

gelatin  is  usually  all  liquefied.  The  deep  colonies,  before  liquefaction 
sets  in,  appear  as  round,  granular  masses  with  scalloped  mari:in>, 
having  a  yellowish-green  color;  the  surface  colonies  have  a  darker 
green  centre,  surrounded  by  a  delicate,  radiating  zone.  In  stick  cul- 
ture* in  gelatin  liquefaction  occurs  at  first  near  the  surface,  in  the  form 
of  a  small  funnel,  and  gradually  extends  downward;  later  the  liquefied 
gelatin  is  separated  from  the  solid  part  of  the  medium  by  a  horizontal 
plane,  a  greenish-yellow  color  being  imparted  to  that  portion  which 
is  in  contact  with  the  air.  On  agar  a  wrinkled,  moist,  greenish-white 
layer  is  developed,  while  the  surrounding  medium  is  bright  green; 
tli is  subsequently  becomes  darker  in  color,  changing  to  blue-green 
or  almost  black.  In  bouillon  the  green  color  is  produced,  and  the 
growth  appears  as  a  delicate,  flocculent  sediment.  Milk  is  coagulated 
with  coincident  acid  reaction. 

There  is  some  difference  of  opinion  in  regard  to  the  pigments 
produced  by  the  bacillus  pyocyaneus.  Gessard's  view  is  that  two  pig- 
ments are  produced  by  this  bacillus — one  of  a  fluorescent  green  and 
the  other  (pyocyanin)  of  a  blue  color.  Pyocyanin  is  soluble  in  chloro- 
form, and  may  be  obtained  from  pure  solution  in  long,  blue  needles. 
The  pigment  which  is  thus  extracted  by  chloroform  distinguishes  the 
bacillus  pyocyaneus  from  other  fluorescing  bacteria. 

Distribution. — This  bacillus  is  very  widely  distributed  in  nature; 
it  is  found  on  the  healthy  skin  of  man,  in  the  feces  of  many  animals,  in 
water  contaminated  by  animal  or  human  material,  in  purulent  dis- 
charges, and  in  serous  wound  secretions. 

Pathogenesis. — Its  presence  in  wounds  greatly  delays  the  process 
of  repair,  and  may  give  rise  to  a  general  depression  of  the  vital  powers 
from  the  absorption  of  its  toxic  products.  Its  pathogenic  effects  on 
animals  have  been  carefully  studied.  It  is  pathogenic  for  guinea-pigs 
and  rabbits.  Subcutaneous  or  intraperitoneal  injections  of  1  c.c.  or 
more  of  a  bouillon  culture  usually  cause  the  death  of  the  animal  in  from 
twenty-four  to  thirty-six  hours.  Subcutaneous  inoculations  produce 
an  extensive  inflammatory  oedema  and  purulent  infiltration  of  the 
tissues;  a  serofibrinous  or  purulent  peritonitis  is  induced  by  the  intro- 
duction of  the  bacillus  into  the  peritoneal  cavity.  The  bacilli  multiply 
in  the  body,  and  may  be  found  in  the  serous  or  purulent  fluid  in  the 
subcutaneous  tissues  or  abdominal  cavity,  as  well  as  in  the  blood  and 
various  organs.  When  smaller  quantities  are  injected  subcutaneously 
the  animal  usually  recovers,  only  a  local  inflammatory  reaction  being 
set  up  (abscess),  and  it  is  subsequently  immune  against  a  second  inocu- 
lation with  doses  which  would  prove  fatal  to  an  unprotected  animal. 
It  is  interesting  to  note  that  Bouchard,  Charrin,  and  GuigBJlld  have 
shown  that  in  rabbits  which  have  been  inoculated  with  a  culture  of 
the  bacillus  anthracis  a  fatal  result  may  be  prevented  by  inoculating 
the  same  animal  soon  after  with  a  pure  culture  of  the  bacillus  pyo- 
cyaneus. Loew  and  Emmerich  have  shown  that  the  en/ymes  pro- 
duced in  the  pyocyaneus  cultures  are  capable  of  destroying  many 
forms  of  bacteria  in  the  test-tube,  and  have  a  slight  protecting  value  in 


376  'BACILLUS  PATHOGENIC  IN  MAN 

the  body.  The  pyocyaneus  bacillus  produces  these  effects  not  only 
through  ferments,  but  by  intracellular  toxins. 

Our  knowledge  of  the  pathogenic  importance  of  the  bacillus  pyo- 
cyaneus in  human  diseases  has  been  much  increased  by  recent  inves- 
tigations. Thus,  cases  have  been  reported  in  which  this  bacillus  has 
been  obtained  in  pure  culture  from  pus  derived  from  the  tympanic 
cavity  in  disease  of  the  middle  ear,  from  cases  of  ophthalmia,  and 
bronchopneumonia.  Kruse  and  Pasquale  have  found  the  same  micro- 
organism in  three  cases  of  idiopathic  abscess  of  the  liver,  in  two  of  them 
in  immense  numbers  and  in  pure  culture.  Ernst  and  Schiirmayer 
report  the  presence  of  the  bacillus  pyocyaneus  in  serous  inflammation 
of  the  pericardial  sac  and  of  the  knee-joint.  Ehlers  gives  the  history  of 
a  disease  in  two  sisters  who  were  attacked  simultaneously  with  fever, 
albuminuria,  and  paralysis.  It  was  thought  that  they  would  prove  to 
be  typhoid  fever  or  meningitis,  but  on  the  twelfth  day  there  was  an 
eruption  of  blisters,  from  the  contents  of  which  the  bacillus  pyocyaneus 
was  isolated.  Jadkewitsch  reports  the  case  of  a  patient  suffering  from 
eczema  of  the  lower  extremities,  in  whom  three  times  during  a  period 
of  ten  years  there  was  eruption  of  boils  containing  blue  pus,  with  accom- 
panying symptoms  of  poisoning,  emaciation,  prostration,  diarrhoea, 
and  paresis.  Krambals  refers  to  seven  cases  in  which  a  general  pyocy- 
aneus infection  occurred,  and  adds  an  eighth  from  his  own  experience. 
In  this  the  bacillus  pyocyaneus  was  obtained  post-mortem  from  green 
pus  in  the  pleural  cavity,  from  serum  in  the  pericardial  sac,  and  from 
the  spleen  in  pure  culture.  Schimmelbusch  states  that  a  physician 
injected  0.5  c.c.  of  sterilized  (by  heat)  culture  into  his  forearm.  As  a 
result  of  this  injection,  after  a  few  hours  he  had  a  slight  chill,  followed 
by  fever,  which  at  the  end  of  twelve  hours  reached  38.8°  C.;  an  erysipe- 
latous-like  swelling  of  the  forearm  occurred,  and  the  glands  in  the 
axilla  were  swollen  and  painful.  Wasserman  reports  an  epidemic  of 
septic  infection  of  the  newborn,  starting  in  the  umbilicus.  In  all  there 
were  eleven  deaths.  Lartigau  found  it  in  well-water,  and  in  great 
abundance  in  the  intestinal  discharges  of  a  number  of  cases  made  ill  by 
drinking  the  water.  It  has  also  been  found  in  a  certain  number  of 
cases  of  gastroenteritis,  where  no  special  cause  of  infection  could  be 
noted. 

We  may,  therefore,  conclude  from  these  facts  that  the  bacillus  pyo- 
cyaneus, although  ordinarily  but  slightly  pathogenic  for  man,  may 
under  certain  conditions  become  a  dangerous  source  of  infection. 
Children  would  seem  to  be  particularly  susceptible. 

The  differential  diagnosis  of  the  pyocyaneus  from  other  fluorescing 
bacteria  is  easy  enough  as  long  as  it  retains  its  pigment-producing 
property.  When  an  agar  culture  is  agitated  with  chloroform  a  blue 
coloration  demonstrates  the  presence  of  this  bacillus.  When  the 
pyocyanin  is  no  longer  formed,  however,  the  diagnosis  is  by  no  means 
easy,  particularly  when  the  pathogenic  properties  are  also  gone. 


BACILLUS  PROTEUS  VULGARIS  377 

Bacillus  Proteus  Vulgaris. 

This  bacillus,  which  is  one  of  the  most  common  and  widely  dis- 
tributed putrefactive  bacteria,  was  discovered  by  Hauser  (1885)  along 
with  other  species  of  proteus  in  putrefying  substances.  These  bacteria 
were  formerly  included  under  the  name  "bacterium  termo"  by  pre- 
vious observers,  who  applied  this  name  to  any  minute  motile  bacilli 
found  in  putrefying  infusions. 

Morphology. — Bacilli  varying  greatly  in  size;  most  commonly  occur- 
ring 0.6/*  broad  and  1.2,«  long,  but  snorter  and  longer  forms  may  also 
be  seen,  even  growing  out  into  flexible  filaments  which  are  sometimes 
more  or  less  wavy  or  twisted  like  braids  of  hair. 

The  bacillus  does  not  form  spores,  and  stains  readily  with  fuchsin  or 
gentian  violet. 

Biology. — An  aerobic,  facultative  anaerobic,  liquefying,  motile  bacillus. 
Grows  rapidly  in  the  usual  culture  media  at  the  room  temperature. 

GROWTH  ox  GELATIN — The  growth  upon  gelatin  plates  containing  5 
per  cent,  of  gelatin  is  very  characteristic.  At  the  end  of  ten  or  twelve 
hours  at  room  temperature  small,  round  depressions  in  the  gelatin  are 
observed,  which  contain  liquefied  gelatin  and  a  whitish  mass  consist- 
ing of  bacilli  in  the  centre.  Under  a  low-power  lens  these  depressions 
are  seen  to  be  surrounded  by  a  radiating  zone  composed  of  two  or 
more  layers,  outside  of  which  is  a  zone  of  a  single  layer,  from  which 
amoeba-like  processes  extend  upon  the  surface  of  the  gelatin.  These 
processes  are  constantly  undergoing  changes  in  their  form  and  posi- 
tion. The  young  colonies  deep  down  in  the  gelatin  are  somewhat 
more  compact,  and  rounded  or  humpbacked;  later  they  are  covered 
with  soft  down;  then  they  form  irregular,  radiating  masses,  and  simu- 
late the  superficial  colonies.  But  it  is  difficult  to  describe  all  the'forms 
which  the  proteus  vulgaris  takes  on  in  all  the  stages  of  its  growth  on 
gelatin  plates.  When  the  consistency  of  the  medium  is  more  solid, 
as  in  10  per  cent,  gelatin,  the  liquefaction  and  migration  of  surface 
colonies  are  more  or  less  retarded.  In  gelatin-stick  cultures  the  growth 
is  less  characteristic — liquefaction  takes  place  rapidly  along  the  line 
of  puncture,  and  soon  the  entire  contents  of  the  tube  are  liquefied. 

Upon  nuirient  agar  a  rapidly  spreading,  moist,  thin,  grayish-white 
layer  appears,  and  migration  of  the  colonies  also  occurs.  Silt  is  coagu- 
lated, with  the  production  of  acid. 

Cultures  in  media  containing  albumin  or  gelatin  have  a  disagree- 
able, putrefactive  odor,  and  become  alkaline  in  reaction.  Growth 
is  most  luxuriant  at  a  temperature  of  24°  C.,  but  is  plentiful  also  at 
37°  C.  It  is  a  facultative  anaerobe  and  grows  also  in  the  absence  of 
oxygen,  but  the  proteus  then  loses  its  power  of  liquefying  gelatin.  It 
produces  indol  and  phenol  from-  peptone  solutions.  The  proteus 
develops  fairly  well  in  urine,  and  decomposes  urea  into  carbonate  of 
ammonia. 

Pathogenesis. — This  bacillus  is  pathogenic  for  rabbits  and  guinea- 
pigs  when  injected  in  large  quantities  into  the  circulation,  the  abdom- 


378  BACTERIA  PATHOGENIC  TO  MAN 

inal  cavity,  or  subcutaneously,  producing  death  of  the  animals  with 
symptoms  of  poisoning.  Hauser  has  obtained  the  bacillus  proteus 
vulgaris  from  a  case  of  purulent  peritonitis,  from  purulent  puerperal 
endometritis,  and  from  a  phlegmonous  inflammation  of  the  hand. 
Brunner  also  reports  similar  infections  in  which  this  organism  wras 
found  associated  with  pus  cocci,  and  Charrin  describes  a  case  of  pleu- 
ritis  during  pregnancy,  in  which  the  proteus  was  present  and  a  foul- 
smelling  secretion  was  produced.  Death  in  this  case,  which  ensued 
without  further  complication,  is  said  to  have  been  due  probably  to  the 
poisonous  products  of  the  proteus. 

An  interesting  example  of  pure  toxaemia  resulting  from  the  toxin 
of  the  proteus  is  reported  by  Levy :  While  conducting  some  experiments 
on  this  organism  he  had  an  opportunity  of  making  a  bacteriological 
examination  in  the  case  of  a  man  who  died  after  a  short  attack  of  cholera 
morbus.  From  the  vomited  material  and  the  stools  he  obtained  a 
pure  culture  of  the  proteus;  but  the  blood,  collected  at  the  autopsy, 
was  sterile.  Tn  the  mean  time  seventeen  other  persons  who  had  eaten 
at  the  same  restaurant  were  taken  sick  in  the  same  way.  Upon  exami- 
nation at  the  restaurant  it  was  found  that  the  bottom  of  the  ice-chest 
in  which  the  meat  was  -kept  was  covered  with  a  slimy,  brown  layer, 
which  gave  off  a  disagreeable  odor.  Cultures  from  this  gave  the  pro- 
teus as  the  principal  organism  present.  Injections  into  animals  of  the 
pure  cultures  produced  similar  symptoms  as  occurred  in  the  human 
subjects. 

Levy  concludes  that  in  so-called  "  flesh  poisoning"  bacteria  of  this 
group  are  chiefly  concerned,  and  that  the  pathogenic  effects  are  due 
to  toxic  products  evolved  during  their  development. 

Booker,  from  his  extended  researches  into  this  subject,  concludes 
that  the  proteus  plays  an  important  part  in  the  production  of  the  mor- 
bid symptoms  which  characterize  cholera  infantum.  Proteus  vulgaris 
was  found  in  the  alvine  discharge  in  a  large  proportion  of  the  cases 
examined  by  him,  but  was  not  found  in  the  feces  of  healthy  infants. 
"The  prominent  symptoms  in  the  cases  of  cholera  infantum  in  which 
the  proteus  bacteria  were  found  were  drowsiness,  stupor,  and  great  re- 
duction in  flesh,  more  or  less  collapse,  frequent  vomiting  and  purging, 
with  watery  and  generally  offensive  stools." 

Next  to  the  bacillus  coli  communis  the  proteus  vulgaris  appears  to 
be  the  micro-organism  most  frequently  concerned  in  the  etiology  of 
pyelonephritis.  In  cases  of  cystitis  and  of  pyelonephritis  this  bacillus 
is  often  found  in  pure  cultures  or  associated  with  other  bacteria.  It 
probably  gets  into  the  bladder  chiefly  through  catheterization.  From 
the  animal  experiments  of  the  authors  above  mentioned,  simple  injec- 
tion of  pure  cultures  of  proteus  into  the  bladder,  without  artificial  sup- 
pression of  urine,  invariably  produces  severe  cystitis.  The  fact  that 
this  organism  grows  in  urine  is  sufficient  to  acconnt  for  the  extension 
of  the  purulent  process  finally  to  the  kidneys. 

The  proteus  vulgaris  is,  however,  a  harmless  parasite  when  located 
in  the  mucous  membrane  of  the  nasal  cavities.  Here  it  only  decom- 


Tin:  (;ROUP  OF  M.\u<;\A\r  <KDI-:M.\  BAC/U.I  379 

poses  the  secretions,  with  the  production  of  a  putrefactive  odor.  On 
the  whole,  considering  the  very  wide  distribution  of  this  organi>ni  in 
nature,  it  is  remarkable  how  few  diseases  are  produced  by  it 

The  Group  of  Malignant  (Edema  Bacilli. 

This  group  is  widely  distributed,  being  found  in  the  superficial  layers 
of  the  soil,  in  putrefying  substances,  in  foul  water,  and  by  invasion 
from  the  intestine,  in  the  blood  of  animals  which  have  been  suffocated. 
One  such  organism  was  discovered  (1877)  by  Pasteur  in  animals 
after  injections  of  putrefying  liquids,  and  named  by  him  "vibrion 
septique."  He  recognized  its  anaerobic  nature,  but  did  not  obtain 
it  in  pure  culture.  Koch  and  Gaffky  (1881)  carefully  studied  this 
micro-organism,  described  it  in  detail,  and  gave  it  the  name  "bacillus 
oedematis  maligni"  (Fig.  115).  This  bacillus  belongs  to  a  group 
which  have  lateral  flagellae,  produce  oval  spores, 
and  grow  only  anaerobically.  FIG.  115 

Morphology.  —  The  oedema  bacillus  is  a  rod  of  S 

from   0.8,«  to  l/^  in  width,  and  of  very  varying  * 

length,  from  2,«  to  10/^  or  more,  according  to  the 


conditions  of   its  cultivation  and  growth.      It   s  /   f 

usually  found   in   pairs,  joined  end   to  end,  but  \        / 

may  occur  in  chains  or  long  filaments.    It  forms 

spores,  and   these  are  situated   in  or  near  the  Bacillus  of  malignant  oedema. 

middle  of  the  body  of  the  rods.     Exceptionally 

the  spores  are  near  the  ends.     The  spores  vary  in  length  and  are  oval 

in  form,  being  often  of  greater  diameter  than  the  bacilli,  to  which  they 

give  a  more  or  less  oval  or  spindle  shape. 

The  bacilli  s/am  readily  by  the  usual  aniline  colors  employed,  but 
are  decolorized  by  Gram's  method. 

Biology.  —  A  strictly  anaerobic,  liquefying,  motile  bacillus.  Forms 
spores.  It  grows  in  all  the  usual  culture  media  in  the  absence  of  oxygen. 
Development  takes  place  at  20°  C.,  but  more  rapidly  and  abundantly 
at  37°  C. 

GROWTH  IN  GELATIN.  —  This  bacillus  may  be  cultivated  in  ordinary 
nutrient  gelatin,  but  the  growth  is  more  abundant  in  glucose  gelatin 
containing  1  or  2  per  cent,  of  glucose.  After  two  to  three  days  small, 
almost  transparent,  circular  colonies  appear  J  to  1  mm.  in  diameter. 
Later,  as  liquefaction  increases,  the  colonies  become  grayish  and  then 
confluent.  Gas  bubbles  are  formed  and  the  gelatin  liquefies. 

GROWTH  ON  AGAR.  —  On  agar  plates  the  colonies  appear  as  dull, 
whitish  points,  irregular  in  outline,  and  when  examined  under  a  low- 
power  lens  are  seen  to  be  composed  of  a  dense  network  of  interlacing 
threads,  radiating  irregularly  from  the  centre  toward  the  periphery 

Blood  serum  is  rapidly  liquefied,  with  the  production  of  gas.  Cultures 
of  the  malignant  oedema  bacillus  give  off  a  peculiar,  diagreealde  odor. 

Pathogenesis.  —  The  bacillus  of  malignant  o-dema  is  especially  patho- 
genic for  mice,  guinea-pigs,  and  rabbits,  although  man,  horses,  dogs, 


380  BACTERIA  PATHOGENIC  TO  MAN 

goats,  sheep,  calves,  pigs,  chickens,  and  pigeons  are  also  susceptible. 
A  small  quantity  of  a  pure  culture  injected  beneath  the  skin  of  a  sus- 
ceptible animal  gives  rise  to  an  extensive  hemorrhagic  oedema  of  the 
subcutaneous  connective  tissue,  which  extends  over  the  entire  surface 
of  the  abdomen  and  thorax,  causing  hypersemia  and  redness  of  the 
superficial  muscles.  No  odor  is  developed,  and  there  is  little,  if  any, 
production  of  gas.  In  infection  with  garden  earth,  owing  to  the  pres- 
ence of  associated  bacilli,  the  effused  serum  is  frothy  from  the  develop- 
ment of  gas,  and  possesses  a  putrefactive  odor.  The  disease,  in  natural 
infection  caused  by  the  contamination  of  wounds  with  earth  or  feces, 
runs  the  course  above  described.  Simple  abrasion  of  the  skin  is  not 
sufficient  to  produce  infection;  owing  to  the  bacillus  being  capable  only 
of  an  anaerobic  existence,  the  poison  must  penetrate  deep  into  the 
tissues.  Malignant  oedema  is  confined  mostly  to  the  domestic  animals, 
but  cases  have  also  been  reported  in  man. 

Animals  which  recover  from  malignant  oedema  are  subsequently 
immune.  Artificial  immunity  may  be  induced  in  guinea-pigs  by  inject- 
ing filtered  cultures  of  the  malignant  oedema  bacillus  in  harmless  quan- 
tities. 

In  man  the  chief  symptom  is  the  sudden  appearance  of  an  oedematous 
swelling  accompanied  by  high  fever.  In  light  cases  this  remains  cir- 
cumscribed; in  severe  cases  it  spreads  widely  and  the  case  ends  fatally. 
Autopsy  shows  a  serous  or  hemorrhagic  infiltration  of  the  subcutaneous 
tissues  and  intramuscular  connective  tissue.  In  the  inflamed  tissue 
the  bacilli  with  and  without  spores  are  found. 

Bacillus  Aerogenes  Capsulatus. 

This  bacillus  was  found  by  Welch  in  the  bloodvessels  of  a  patient 
suffering  with  aortic  aneurysm ;  on  autopsy,  made  in  cool  weather,  eight 
hours  after  death,  the  vessels  were  observed  to  be  full  of  gas  bubbles. 
Since  then  it  has  been  found  in  a  number  of  cases  in  which  gas 
has  developed  from  within  sixty  hours  of  death  until  some  hours  after 
death.  These  cases  are,  as  a  rule,  marked  by  delirium,  rapid  pulse, 
high  temperature,  and  the  development  of  emphysema  and  discolora- 
tion of  the  diseased  area,  or  of  marked  abdominal  distention  when  the 
peritoneal  cavity  is  involved. 

Morphology. — Straight  or  slightly  curved  rods,  with  rounded  or 
sometimes  square-cut  ends;  somewhat  thicker  than  the  anthrax  bacilli 
and  varying  in  length;  occasionally  long  threads  and  chains  are  seen. 
The  bacilli  in  the  animal  body,  and  sometimes  in  cultures,  are  enclosed 
in  a  transparent  capsule. 

Biology. — An  anaerobic,  non-motile,  non-liquefying  bacillus.  Does 
not  form  spores.  Growth  is  rapid  at  37°  C.,  in  the  usual  culture  media 
in  the  absence  of  oxygen,  and  is  accompanied  by  the  production  of 
gas.  Nutrient  gelatin  is  not  liquefied  by  the  growth  of  this  bacillus, 
but  it  is  gradually  peptonized.  In  agar  colonies  "are  developed  which 
are  from  1  to  2  mrn.  or  more  in  diameter,  grayish-white  in  color,  and 


BACILLUS  AEROGENES  CAPSULATUS  381 

in  the  form  of  flattened  spheres,  ovals,  or  irregular  masses,  beset  with 
hair-like  projections.  Bouillon  is  diffusely  clouded,  and  a  white  sedi- 
ment is  formed.  Milk  is  rapidly  coagulated. 

Pathogenesis. — Usually  non-pathogenic  in  healthy  animals,  although 
Dunham  found  that  the  bacillus  taken  freshly  from  human  infection 
is  sometimes  very  virulent.  When  quantities  up  to  2.5  c.c.  of  fresh 
bouillon  cultures  are  injected  into  the  circulation  of  rabbits  and  the 
animals  killed  shortly  after  the  injection,  the  bacilli  develop  rapidly, 
with  an  abundant  formation  of  gas  in  the  bloodvessels  and  organs, 
especially  the  liver.  The  following  is  one  of  the  best  methods  of  obtain- 
ing the  bacilli :  The  material  suspected  to  contain  the  bacillus  alone 
or  associated  with  other  bacteria  is  injected  into  rabbits,  which  are 
killed,  kept  at  37°  C.,  and  cultures  made  twenty-four  hours  later  from 
their  bodies. 

It  is  suggested  by  Welch  that  in  some  of  the  cases  in  which  death 
has  been  attributed  to  the  entrance  of  air  into  the  veins  the  gas  found 
at  the  autopsy  may  not  have  been  atmospheric  air,  but  may  have  been 
produced  by  this  or  some  similar  micro-organism  entering  the  circula- 
tion and  developing  shortly  before  and  after  death.  The  bacillus  has 
been  found  in  the  dust  of  hospitals. 


CHAPTER    XXIX. 

THE  ANTHRAX  BACILLUS  AND  THE  BACILLUS  OF  SYMPTOMATIC 

ANTHRAX. 

Bacillus  Anthracis. 

ANTHRAX  is  an  acute  infectious  disease  which  is  very  prevalent 
among  animals,  particularly  sheep  and  cattle.  Geographically  and 
zoologically  it  is  the  most  widespread  of  all  infectious  disorders.  It 
is  much  more  common  in  Europe  and  in  Asia  than  in  America.  The 
ravages  among  herds  of  cattle  in  Russia  and  Siberia,  and  among  sheep 
in  certain  parts  of  France,  Hungary,  Germany,  Persia,  and  India 
are  not  equalled  by  any  other  animal  plague.  Local  epidemics  have 
occasionally  occurred  in  England,  where  it  is  known  as  splenic  fever. 
In  this  country  the  disease  is  rare.  In  infected  districts  the  greatest 
losses  are  incurred  during  the  hot  months  of  summer. 

The  disease  also  occurs  in  man  as  the  result  of  infection,  either 
through  the  skin,  the  intestines,  or  in  rare  instances  through  the  lungs. 
It  is  found  in  persons  whose  occupations  bring  them  into  contact  with 
animals  or  animal  products,  as  stablemen,  shepherds,  tanners,  butchers, 
and  those  who  work  in  wool  and  hair.  Two  forms  of  the  disease  have 
been  described — the  external  anthrax,  or  malignant  pustules,  and  the 
internal  anthrax,  of  which  there  are  intestinal  and  pulmonary  forms, 
the  latter  being  known  as  "wool-sorters'  disease." 

Owing  to  the  fact  that  anthrax  was  the  first  infectious  disease  which 
was  shown  to  be  caused  by  a  specific  micro-organism,  and  to  the  close 
study  which  it  received  in  consequence,  this  disease  has  probably  con- 
tributed more  to  our  general  knowledge  of  bacteriology  than  any  other 
infectious  malady. 

Pollender  in  1849  observed  that  the  blood  of  animals  suffering  from 
splenic  fever  always  contained  minute  rod-shaped  bacteria.  Davaine 
in  1863  announced  to  the  French  Academy  of  Sciences  the  results  of 
his  inoculation  experiments,  and  asserted  the  etiological  relations  of 
the  micro-organism  to  the  disease,  \vith  which  his  investigation  showed 
it  to  be  constantly  associated.  For  a  long  time  this  conclusion  was 
energetically  opposed  until,  in  1879,  Pasteur,  Koch,  and  others  estab- 
lished its  truth  by  obtaining  the  bacillus  in  pure  cultures,  and  showing 
that  the  inoculation  of  these  cultures  produced  anthrax  in  susceptible 
animals  as  certainly  as  did  the  blood  of  an  animal  recently  dead  from 
the  disease. 

Morphology. — Slender,  cylindrical,  non-motile  rods,  having  a  breadth  of 
I/*  to  1.25/>«,  and  ranging  from  2//  or  3/J.  to  20//  or  25/>«  in  length.  Some: 


THE  .\\THRAX  BACILLUS  333 

times  short,  isolated  rods  are  seen,  and,  again,  shorter  or  longer  chains 
or  threads  made  up  of  several  rods  joined  end  to  end.  In  suitable 
culture  media  very  long,  flexible  filaments  may  be  observed,  which 
are  frequently  united  in  twisted  or  plaited  cord-like  bundles.  (See 
Fig.  116  and  Fig.  13,  p.  29,  and  Fig.  17,  p.  33.)  These  filaments  in 
hanging-drop  cultures,  before  the  development  of  spores,  appear  to 
be  homogeneous  or  nearly  so;  but  in  stained  preparations  they  are 
seen  to  be  composed  of  a  series  of  rectangular,  deeply  stained  segments. 
When  obtained  directly  from  the  blood  of  an  infected  animal  the  free 
ends  of  the  rods  are  slightly  rounded,  but  those  coming  in  contact  with 
one  another  are  quite  square.  In  cultures  the  ends  are  seen  to  be  a 
trifle  thicker  than  the  body  of  the  cell  and  somewhat  concave,  giving 
the  appearance  of  joints  of  bamboo.  At  one  time  much  stress  was 
laid  upon  these  peculiarities  as  distinguishing  marks  of  the  anthrax 


FIG. lie 


Anthrax  bacillus.    X  900  diameters.    Agar  culture. 

bacillus;  but  it  has  been  found  that  they  are  the  effects  of  artificial 
cultivation  and  not  necessarily  characteristic  of  the  organism  under 
all  conditions.  Another  peculiarity  of  this  bacillus  is  that  it  is  enclosed 
in  a  transparent  envelope  or  capsule,  which  in  stained  preparations 
may  be  distinguished  by  its  taking  on  a  lighter  stain  than  the  deeply 
stained  rods  which  it  surrounds. 

Under  favorable  conditions  in  cultures  spores  are  developed  in  the 
bacilli.  These  spores  are  elliptical  in  shape  and  about  one  and  a  half 
times  longer  than  broad.  They  first  appear  as  small,  refractive  granules 
distributed  at  regular  intervals,  one  in  each  rod.  As  the  spore  develops 
the  mother-cell  becomes  less  and  less  distinct,  until  it  disappears  alto- 
gether, the  complete  oval  spore  being  set  free  by  its  dissolution.  (See 
Fig.  117,  Fig.  13,  p.  29,  and  Fig.  17,  p.  33.)  Irregular  sponilation 
sometimes  takes  place,  and  occasionally  there  is  no  spore  formation, 
as  in  varieties  of  non-spore-bearing  anthrax. 


384  BACTERIA  PATHOGENIC  TO  MAN 

Staining. — The  anthrax  bacillus  stains  readily  with  all  the  aniline 
colors,  and  also  by  Gram's  method,  when  not  left  too  long  in  the 
decolorizing  solution.  In  sections  good  results  may  be  obtained  by 
the  employment  of  Gram's  solution  in  combination  with  carmine, 
but  when  only  a  few  bacilli  are  present  this  method  is  not  always  reli- 
able, as  some  of  the  bacilli  are  generally  decolorized. 

Biology. — The  anthrax  bacillus  grows  easily  in  a  variety  of  nutrient 
media  at  a  temperature  from  18°  to  43°  C.,  37°  C.  being  the  most  favor- 
able temperature.  Under  12°  C.  no  development  takes  place,  as  a  rule, 
though  by  gradually  accustoming  the  bacillus  to  a  lower  temperature 
it  may  be  induced  to  grow  under  these  conditions.  Under  14°  C.  and 
above  43°  C.  spore  formation  ceases.  The  lower  limit  of  growth  and 
of  sporulation  is  of  practical  significance  in  determining  the  question 
whether  development  can  occur  in  the  bodies  of  animals  dead  from 
anthrax  when  buried  at  certain  depths  in  the  earth.  Kitasato  has 

FIG. 117 


Spores  heavily  stained  (in  specimen  red).    Bodies  of  disintegrating  bacilli  faintly  stained 
(in  specimen  blue).    X  1000  diameters 

shown  that  at  a  depth  of  1.5  metres  the  earth  in  July  has  a  tempera- 
ture of  15°  C.  at  most,  and  that  under  these  conditions  a  scanty  sporu- 
lation of  anthrax  bacilli  is  possible,  but  that  at  a  depth  of  2  metres 
sporulation  no  longer  occurs.  The  anthrax  bacillus  is  aerobic — that 
is,  its  growth  is  considerably  enhanced  by  the  presence  of  oxygen — 
but  it  grows  also  under  anaerobic  conditions,  as  is  shown  by  its  growth 
at  the  bottom  of  the  line  of  puncture  in  stick  cultures  in  solid  media; 
but  under  these  conditions  it  no  longer  produces  the  peptonizing  fer- 
ment which  it  does  with  free  access  of  air.  Furthermore,  the  presence 
of  oxygen  is  absolutely  necessary  for  the  formation  of  spores,  while 
carbonic  acid  gas  retards  sporulation.  This  explains,  perhaps,  why 
sporulation  does  not  take  place  within  the  animal  body  either  before 
or  after  death. 

This  bacillus  grows  best  in  neutral  or  slightly  alkaline  media.     It 
may  be  cultivated  in  infusions  of  meat  or  of  various  vegetables,  in 


777 /•:  .l.\  •/•///,'. l.V   BACILLUS  385 

urine,  etc.,  provided  the  reaction  be  not  decidedly  acid,  which  arrests 
development.  It  grows  in  cow-dung  and  in  more  or  less  contaminated 
earth.  It  is  also  capable  of  leading  a  saprophytic  existence.  The 
bacillus  is  non-motile. 

GROWTH  IN  GELATIN. — In  gelatin-plate  cultures,  at  the  end  of  twenty- 
four  to  thirty-six  hours  at  24°  C.,  small,  white,  opaque  colonies  are 
developed,  which,  under  a  low-power  lens,  are  seen  to  be  dark  gray  in 
the  centre  and  surrounded  by  a  greenish,  irregular  border,  made  up  of 
wavy  filaments.  As  the  colony  develops  on  the  surface  of  the  gelatin 
these  wavy  filaments  spread  out,  until  finally  the  entire  colony  consists 
of  a  light-gray,  tangled  mass,  which  has  been  likened  to  a  Medusa  head 
(Fig.  118). 

At  the  same  time  the  gelatin  begins  to  liquefy,  and  the  colony  is  soon 
surrounded  by  the  liquefied  medium,  upon  the  surface  of  which  it  floats 

FIG.  118 


Colonies  of  bacillus  anthracis  upon  gelatin  plates :  a,  at  the  end  of  twenty-four  hours  ; 
6,  at  the  end  of  forty-eight  hours.    X  80.    (F.  Fliigge.) 

as  an  irregular,  white  pellicle.  In  gelatin-stick  cultures  at  first  develop- 
ment occurs  along  the  line  of  puncture  as  a  delicate  white  thread,  from 
which  irregular,  hair-like  projections  soon  extend  perpendicularly  into 
the  culture  medium,  the  growth  being  most  luxuriant  near  the  surface, 
but  continuing  also  below.  At  the  end  of  two  or  three  days  liquefaction 
of  the  medium  commences  at  the  surface  and  gradually  progresses 
downward. 

GROWTH  <>\  A<;AR. — The  growth  on  (if/nr-ftlafr  cultures  in  the  incu- 
bator at  37°  0\  is  similar  to  that  on  gelatin,  and  is  still  more  character- 
i>ti<-  and  beautiful  in  appearance.  A  grayish-white  layer  i>  formed  on 
the  surface  within  twenty-four  hours,  which  ^pread-  rapidly  and  is 
seen  to  be  made  up  of  interlaced  threads. 

GROWTH  IN  HOIII.I.ON. — The  growth  is  characterized  by  the  forma- 
tion of  flaky  masses,  which  sink  as  a  sediment  to  the  bottom  of  the 
tube,  leaving  the  supernatant  liquid  clear. 

25 


/ 


386  BACTERIA  PATHOGENIC  TO  MAN 

Spore  formation,  as  already  noted,  only  takes  place  in  the  pres- 
ence of  oxygen,  and  at  a  temperature  of  15°  to  43°  C.  There  is  no 
development  of  spores  at  a  greater  depth  than  1.5  metres  in  the  earth, 
or  in  the  bodies  of  living  or  dead  animals;  but  spores  may  be  found 
in  the  fluids  containing  the  bacilli  when  these  come  in  contact  with 
the  air,  as  in  bloody  discharges  from  the  nostrils  or  from  the  bowels 
of  the  dead  animal. 

There  are  certain  non-spore  bearing  species  of  anthrax.  Sporeless 
varieties  have  also  been  produced  artificially  by  cultivating  the  typical 
anthrax  bacillus  under  unfavorable  conditions,  among  which  may 
be  mentioned  the  addition  of  antiseptics,  as  carbolic  acid.  Varieties 
differing  in  their  pathogenic  power  may  also  be  produced  artificially. 
Pasteur  produced  an  "attenuated  virus"  by  keeping  his  cultures  for  a 
considerable  time  before  replanting  them  upon  fresh  soil. 

Anthrax  cultures  containing  spores  retain  their  vitality  for  years; 
in  the  absence  of  spores  the  vitality  is  much  more  rapidly  lost.  When 
grown  in  liquids  rich  in  albumin  the  bacilli  attain  a  considerable  degree 
of  resistance;  thus  dried  anthrax  blood  has  been  found  to  retain  its 
virulence  for  sixty  days,  while  dried  bouillon  cultures  only  did  so  for 
twenty-one  days.  Dried  anthrax  spores  may  be  preserved  for  many 
years  without  losing  their  vitality  or  virulence.  They  also  resist  a 
'I comparatively  high  temperature.  Exposed  in  dry  air  they  require  a 
temperature  of  140°  C.  maintained  for  three  hours  to  destroy  them; 
but  suspended  in  a  liquid  they  are  destroyed  in  four  minutes  by  a  tem- 
perature of  100°  C. 

Pathogenesis. — The  anthrax  bacillus  is  pathogenic  for  cattle,  sheep, 
(except  the  Algerian  race),  horses,  swine,  mice,  guinea-pigs,  and  rabbits. 
Rats,  cats,  dogs,  chickens,  owls,  pigeons,  and  frogs  are  but  little  sus- 
ceptible to  infection.  Small  birds — the  sparrow  particularly — are 
somewhat  susceptible.  Man,  though  subject  to  local  infection  and 
occasionally  to  internal  forms  of  the  disease,  is  not  as  susceptible  as 
some  of  the  lower  animals. 

In  susceptible  animals  the  anthrax  bacillus  produces  a  true  septi- 
caemia. Among  test  animals  mice  are  the  most  susceptible,  succumb- 
ing to  very  minute  injections  of  a  slightly  virulent  virus;  next  guinea- 
pigs,  and  lastly  rabbits,  both  of  these  animals  dying  after  inoculation 
with  virulent  bacilli.  Infection  is  most  promptly  produced  by  intro- 
duction of  the  bacilli  into  the  circulation  or  the  tissues,  but  inocu- 
lation by  contact  with  wounds  on  the  skin  also  causes  infection.  It  is 
difficult  to  produce  infection  by  the  ingestion  even  of  spores;  but  it 
may  readily  be  caused  by  inhalation,  particularly  of  spores. 

Subcutaneous  injections  of  these  susceptible  animals  results  in 
^ath  in  from  one  to  three  days.  Comparatively  little  local  reaction 
occurs  immediately  at  the  point  of  inoculation,  but  beyond  this  there 
is  an  extensive  oedema  of  the  tissues.  Very  few  bacilli  are  found  in 
the  blood  in  the  larger  vessels,  but  in  the  internal  organs,  and  especially 
in  the  capillaries  of  the  liver,  the  kidneys,  and  the  lungs,  they  are  present 
in  great  numbers.  In  some  places,  as  in  the  glomeruli  of  the  kidneys, 


THE  ANTHRAX  BACILLUS  387 

the  capillaries  will  be  seen  to  be  stuffed  full  of  bacilli,  and  hemor- 
rhages, probably  due  to  rupture  of  capillaries  by  the  mechanical  pressure 
of  the  bacilli  which  are  developing  within  them,  may  occur.  The  patho- 
logical lesions  in  animals  infected  by  anthrax  are  not  marked  except 
in  the  spleen,  which,  as  in  other  forms  of  septicaemia,  is  greatly  enlarged. 

Occurrence  in  Cattle  and  Sheep. — Cattle  and  sheep  are  affected  chiefly 
with  the  intestinal  form  of  anthrax,  infection  in  these  animals  com- 
monly resulting  from  the  ingestion  of  food  containing  spores.  The 
bacillus  itself,  in  the  absence  of  spores,  is  quickly  destroyed  by  the 
gastric  juice.  The  disease  usually  takes  a  rapid  course,  and  the  mor- 
tality is  high — 70  to  80  per  cent.  The  pathological  lesions  consist  of 
numerous  ecchymoses,  enlargement  of  the  lymphatic  glands,  serous, 
fatty,  and  hemorrhagic  infiltration  of  the  mediastinum  and  mesentery, 
of  the  mucous  membranes  of  the  pharynx  and  larynx,  and  particularly 
of  the  duodenum,  great  enlargement  of  the  spleen,  and  parenchymatous 
changes  in  the  lymphatic  organs.  The  blood  is  very  dark  and  tar-like. 
Bacilli  are  present  in  enormous  masses. 

Sheep  are  also  subject  to  external  anthrax,  infection  taking  place 
by  way  of  the  skin ;  cattle  are  seldom  infected  in  this  way.  At  the  point 
of  inoculation  there  develops  a  hard,  circumscribed  boil — the  so- 
called  anthrax  carbuncle;  or  there  may  be  diffuse  oedema,  with  great 
swelling  of  the  parts.  When  death  occurs  the  appearances  are  similar 
to  those  in  intestinal  anthrax,  except  that  the  duodenum  is  usually 
less  affected;  but  in  all  cases  metastasis,  occurs  in  various  parts  of  the 
body,  brought  about,  no  doubt,  by  previous  hemorrhages. 

Occurrence  in  Man. — The  disease  does  not  occur  spontaneously  in 
man,  but  always  results  from  infection,  either  through  the  skin,  the 
intestines,  or  occasionally  by  inhalation  through  the  lungs.  It  is  usually 
produced  by  cutaneous  infection  through  inoculation  of  exposed  sur- 
faces— the  hands,  arms,  or  face.  Infection  of  the  face  or  neck  would 
seem  to  be  the  most  dangerous,  the  mortality  in  such  cases  being  26 
per  cent.;  while  infection  of  the  extremities  is  rarely  fatal. 

External  anthrax  in  man  is  similar  to  this  form  of  the  disease  in 
animals.  There  are  two  forms:  malignant  pustule  or  carbuncle,  and, 
less  commonly,  malignant  anthrax  oedema. 

In  malignant  pustule,  at  the  site  of  inoculations,  a  small  papule 
develops,  which  becomes  vesicular.  Inflammatory  induration  extends 
around  this,  and  within  thirty-six  hours  there  is  a  dark-brownish  eschar 
in  the  centre,  at  a  little  distance  from  which  there  may  be  a  series  of 
small  vesicles.  The  brawny  induration  may  be  extreme.  There  may 
also  be  considerable  oedema  of  the  parts.  In  most  cases  there  is  no  fever; 
or  the  temperature  at  first  rises  rapidly  and  the  febrile  phenomena  are 
marked.  Death  may  take  place  in  from  three  to  five  days.  In  cases 
which  recover  the  symptoms  are  slighter.  In  the  mildest  form  there 
may  be  only  slight  swelling. 

Malignant  anthrax  oedema  occurs  in  the  eyelids,  and  also  in  the  head 
and  neck,  sometimes  the  hand  and  arm.  It  is  characterized  by  the 
absence  of  the  papule  and  vesicle  forms,  and  by  the  most  extensive 


388  BACTERIA  PATHOGENIC  TO  MAN 

oedema.  The  oedema  may  become  so  intense  that  gangrene  results; 
such  cases  usually  prove  fatal. 

The  bacilli  are  found  on  microscopic  examination  of  the  fluid 
from  the  pustule  shortly  after  infection;  later  the  typical  anthrax  bacilli 
are  often  replaced  by  involution  forms.  In  this  case  resort  may  be 
had  to  cultures,  animal  inoculation,  or  examination  of  sections  of  the 
extirpated  tumor.  The  bacilli  are  not  present  in  the  blood  until  just 
before  death.  Along  with  the  anthrax  bacilli  pus  cocci  are  often  found 
in  the  pustule  penetrating  into  the  dead  tissue. 

Internal  anthrax  is  much  less  common  in  man;  it  does,  however, 
occur  now  and  then.  There  are  two  forms  of  this:  the  intestinal  form, 
or  mycosis  intestinalis,  and  the  pulmonic  form,  or  wool-sorters'  dis- 
ease. 

Intestinal  anthrax  is  caused  by  infection  through  the  stomach  and 
intestines,  and  results  probably  from  the  eating  of  raw  flesh  or  un- 
boiled milk  of  diseased  animals.  That  the  eating  of  flesh  from  infected 
animals  is  comparatively  harmless  is  shown  by  Gerlier,  who  states  that 
of  400  persons  who  were  known  to  have  eaten  such  meat  not  one  was 
affected  with  anthrax.  On  the  other  hand,  an  epidemic  of  anthrax 
was  produced  among  wild  animals,  according  to  Jansen,  by  feeding 
them  on  infected  horse-flesh.  It  is  evident,  therefore,  that  there  is  a 
possibility  of  infection  being  caused  in  this  way.  The  recorded  cases 
of  intestinal  anthrax  in  man  have  occurred  in  persons  who  were  in 
the  habit  of  handling  hides,  hair,  etc.,  which  were  contaminated  with 
spores;  in  those  who  were  conducting  laboratory  experiments,  and  rarely 
it  has  been  produced  by  the  ingestion  of  food,  such  as  raw  ham  and 
milk.  The  symptoms  produced  in  this  disease  are  those  of  intense 
poisoning:  chill,  followed  by  vomiting,  diarrhoea,  moderate  fever,  and 
pains  in  the  legs  and  back.  The  pathological  lesions  are  similar  to 
those  described  in  animals. 

Wool-sorter  ft'  disease,  or  pulmonic  anthrax,  is  found  in  large  estab- 
lishments in  which  wool  and  hair  are  sorted  and  cleansed,  and  is  caused 
by  the  inhalation  of  dust  contaminated  with  anthrax  spores.  The 
attack  comes  on  with  chills,  prostration,  then  fever.  The  breathing  is 
rapid,  and  the  patient  complains  of  pain  in  the  chest.  There  may  be 
a  cough  and  signs  of  bronchitis.  The  bronchial  symptoms  in  some 
instances  are  pronounced.  Death  may  occur  in  from  two  to  seven 
days.  The  pathological  changes  produced  are  swelling  of  the  glands 
of  the  neck,  the  formation  of  foci  of  necrosis  in  the  air  passages,  oedema 
of  the  lungs,  pleurisy,  bronchitis,  enlargement  of  the  spleen,  and  paren- 
chymatous  degenerations. 

Prophylaxis  against  Anthrax  Infection. — Numerous  investigations 
have  been  undertaken  with  the  object  of  preventing  infection  from 
anthrax.  The  efforts  of  Pasteur  to  effect  immunity  in  animals  by 
preventive  inoculations  of  "attenuated  virus"  of  the  anthrax  bacillus, 
opened  a  new  field  of  productive  original  research.  Following  in  his 
wake  many  others  have  devised  methods  of  immunization  against 
anthrax  infection;  but  the  one  adopted  by  Pasteur,  Chamberland,  arid 


////.  BACILLUS  OF  SYMPTOMATIC  ANTHRAX  389 

Roux  lias  alone  been  practically  employed  on  a  large  scale.  According 
to  these  authors,  two  anthrax  cultures  of  different  degrees  of  virulence 
attenuated  by  cultivation  at  42°  to  43°  C.,  are  used  for  inoculation. 
Vaccine  No.  1  kills  mice,  but  not  guinea-pigs;  vaccine  No.  2  kills  guinea- 
pigs,  but  not  rabbits,  according  to  Koch,  Gaffky,  and  Loeffler.  The 
animals  to  be  inoculated — viz.,  sheep  and  cattle — are  first  given  a  sub- 
cutaneous injection  of  one  to  several  tenths  of  a  cubic  centimetre  of  a 
four-day-old  bouillon  culture  of  Vaccine  No.  1 ;  after  ten  to  twelve  days 
they  receive  a  similar  dose  of  Vaccine  No.  2.  Prophylactic  inoculations 
given  in  this  way  have  been  widely  employed  in  France,  Hungary, 
and  Russia,  with  apparently  good  results. 

Bacterial  Cultures  for  Diagnosis. — The  detection  of  the  anthrax 
bacillus  is  ordinarily  not  difficult,  as  this  organism  presents  morpho- 
logical, biological,  and  pathogenic  characteristics  which  distinguish  it 
from  all  other  bacteria.  In  the  later  stages  of  the  disease,  however, 
the  bacilli  may  be  absent  or  difficult  to  find,  and  cultivation  on  artificial 
media  and  experimental  inoculation  in  animals  are  not  always  fol- 
lowed by  positive  results.  Even  in  sections  taken  from  the  extirpated 
pustule  it  is  sometimes  difficult  to  detect  the  bacilli.  In  such  cases 
only  a  probable  diagnosis  of  anthrax  can  be  made.  It  should  be  remem- 
bered that  the  bacilli  are  not  found  in  the  blood  until  shortly  before 
death,  and  then  only  in  varying  quantity;  thus  blood  examinations 
often  give  negative  results,  though  the  bacilli  may  be  present  in  large 
numbers  in  the  spleen,  kidneys,  and  other  organs  of  the  body.  The 
suspected  material  should  be  streaked  over  nutrient  agar  in  Petri  plates 
and  inoculated  in  mice. 

Differential  Diagnosis. — Among  other  bacteria  which  may  possibly  be 
mistaken  for  anthrax  bacilli  are  the  bacillus  subtilis  and  the  bacillus 
of  malignant  oedema.  The  former  is  distinguished  by  its  motility,  by 
various  cultural  peculiarities,  and  by  being  non-pathogenic.  The 
latter  differs  from  the  anthrax  bacillus  in  form  and  motility,  in  being 
decolorized  by  Gram's  solution,  in  being  a  strict  anaerobe,  and  in 
various  pathogenic  properties. 

The  diagnosis  of  internal  anthrax  in  man  is  by  no  means  easy,  unless 
the  history  points  definitely  to  infection  in  the  occupation  of  the  indi- 
vidual. In  cases  of  doubt  cultures  should  be  made  and  inoculations 
performed  in  animals.  According  to  Cornil  and  Babes,  some  of  these 
cases  may  possibly  be  caused  by  organisms  other  than  the  bacillus  of 
anthrax. 

Bacillus  Anthracis  Symptomatic!  (Bacillus  of  Symptomatic 

Anthrax). 

Like  the  bacilli  of  anthrax  and  of  malignant  oedema,  both  of  which 
it  resembles  in  other  respects  also,  the  bacillus  of  symptomatic  anthrax 
is  an  inhabitant  of  the  soil.  It  is  found  as  the  chief  cause  of  the  dis- 
ease in  animals — principally  cattle  and  sheep — known  as  "black  leg," 
"quarter  evil,"  or  symptomatic  anthrax  (rausch  brand,  German;  charbon 
symptomatic  pie,  French),  a  disease  which  prevails  in  certain  localities 


390  BACTERIA  PATHOGENIC  TO  MAN 

in  summer,  and  is  characterized  by  a  peculiar  emphysematous  swelling 
of  the  subcutaneous  tissues  and  muscles,  especially  over  the  quarters. 

Morphology. — Bacilli  having  rounded  ends,  from  0.5/^  to  0.6/>«  broad 
and  from  3f*  to  5/^  long;  mostly  isolated;  also  occurring  in  pairs,  joined 
end-to-end,  but  never  growing  out  into  long  filaments,  as  the  anthrax 
bacilli  in  culture  and  the  bacilli  of  malignant  oedema  in  the  bodies 
of  animals  are  frequently  seen  to  do.  In  the  hanging  drop  the  bacilli 
are  observed  to  be  actively  motile,  and  in  stained  preparations  flagella 
may  be  demonstrated  surrounding  the  periphery.  The  spores  are 
elliptical  in  shape,  usually  thicker  than  the  bacilli,  lying  near  the  middle 
of  the  rods,  but  rather  toward  one  extremity.  This  gives  to  the  bacilli 
containing  spores  a  somewhat  spindle  shape. 

Stains  with  the  ordinary  aniline  dyes,  but  not  with  Gram's  method, 
or  only  with  difficulty  and  after  long  treatment  or  intense  colors. 


FIG.  119 


jf V    '   -\ 

.       I '  '  \  ,. 

'« ..-v,  *H 

.1,1       //  / 

1  •»'  '} !  u 

•  /  v  ^ 

Bacilli  of  symptomatic  anthrax,  showing  spores.    (After  Zettnow.) 

Biology. — Like  the  bacillus  of  malignant  oedema,  this  is  a  strict 
anaerobe,  and  cannot  be  cultivated  in  an  atmosphere  in  which  oxygen 
is  present.  It  grows  best  under  hydrogen,  and  does  not  grow  under 
carbonic  acid.  This  bacillus  develops  at  the  room  temperature  in 
the  usual  culture  media,  in  the  absence  of  oxygen,  but  it  grows  best 
in  those  to  which  1.5  to  2  per  cent,  of  glucose  or  5  per  cent,  of  glycerin 
has  been  added. 

GROWTH  ON  AGAR. — The  colonies  on  agar  are  somewhat  more  com- 
pact than  those  of  malignant  oedema,  but  they  also  send  out  projec- 
tions very  often.  In  agar-stick  cultures,  in  the  incubator,  growth  occurs 
after  a  day  or  two  also  some  distance  below  the  surface,  and  is  accom- 
panied by  the  production  of  gas  and  a  peculiar  disagreeable  acid  odor. 

Pathogenesis. — The  bacillus  of  symptomatic  anthrax  is  pathogenic 
for  cattle  (which  are  immune  against  malignant  oedema),  sheep,  goats, 
guinea-pigs,  and  mice;  horses,  asses,  and  white  rats,  when  inoculated 
with  a  culture  of  this  bacillus,  present  only  a  limited  reaction;  and 
rabbits,  swine,  dogs,  cats,  chickens,  ducks,  and  pigeons  are,  as  a  rule, 


THE  BACILLUS  OF  SYMPTOMATIC  ANTHRAX  391 

naturally  immune  to  the  disease.  -  The  guinea-pig  is  the  most  suscept- 
ible of  test  animals.  When  susceptible  animals  are  inoculated  subcu- 
taneously  with  pure  cultures  of  this  organism,  or  with  spores  attached 
to  a  silk  thread,  or  with  bits  of  tissue  from  the  affected  parts  of  another 
animal  dead  of  the  disease,  death  ensues  in  from  twenty-four  to  thirty- 
six  hours.  At  the  autopsy  a  bloody  serum  is  found  in  the  subcutaneous 
tissues,  extending  from  the  point  of  inoculation  over  the  entire  surface 
of  the  abdomen,  and  the  muscles  present  a  dark-red  or  black  appear- 
ance, even  more  intense  in  color  than  in  malignant  oedema,  and  there 
is  a  considerable  development  of  gas.  The  lymphatic  glands  are  mark- 
edly hypersemic. 

The  disease  occurs  chiefly  in  cattle,  more  rarely  in  sheep  and  goats; 
horses  are  not  attacked  spontaneously — i.  e.,  by  accidental  infection. 
In  man  infection  has  never  been  produced,  though  ample  opportunity 
by  infection  through  wounds  in  slaughter-houses  and  by  ingestion  of 
infected  meat  has  been  given.  The  usual  mode  of  natural  infection 
by  symptomatic  anthrax  is  through  wounds  which  penetrate  not  only 
the  skin,  but  the  deep,  intercellular  tissues;  some  cases  of  infection  by 
ingestion  have  been  observed.  The  pathological  findings  present  the 
conditions  above  described  as  occurring  in  experimental  infection. 

DISTRIBUTION  OUTSIDE  OF  THE  BODY. — Symptomatic  anthrax,  like 
anthrax  and  malignant  oedema,  is  a  disease  of  the  soil,  but  it  shows  a 
more  limited  endemic  distribution  than  the  former,  and  is  differently 
distributed  over  the  earth's  surface  than  the  second  of  these  diseases, 
being  confined  especially  to  places  over  which  infected  herds  of  cattle 
have  been  pastured.  It  is  doubtful  whether  the  bacilli  are  capable 
of  development  outside  of  the  body  like  anthrax.  In  the  form  of  spores, 
however,  reproduction  may  take  place;  and  by  contamination  with 
these,  through  deep  wounds  acquired  by  animals  in  infected  pastures, 
the  disease  is  spread. 

TOXINS. — Under  favorable  conditions  extracellular  toxins  are  formed 
so  that  the  filtrate  of  cultures  is  very  poisonous.  Injections  of  the 
toxin  into  animals  excite  the  production  of  antitoxins. 

To  recapitulate  briefly,  the  principal  points  for  differentiating  this 
bacillus  from  the  bacillus  of  malignant  oedema,  which  it  closely  resem- 
bles, are:  it  is  smaller;  it  does  not  develop  into  long  threads  in  the 
tissues;  it  is  more  actively  motile,  and  forms  spores  more  readily  in  the 
animal  body  than  does  the  bacillus  of  malignant  oedema.  It  is  patho- 
genic for  cattle,  while  malignant  oedema  is  not;  and  swine,  dogs,  rabbits, 
chickens,  and  pigeons,  which  are  readily  infected  with  malignant  oedema, 
are  not,  as  a  rule,  susceptible  to  symptomatic  anthrax. 

PREVENTIVE  INOCULATIONS. — It  is  well  known  to  veterinarians  that 
recovery  from  one  attack  of  symptomatic  anthrax  protects  an  animal 
against  a  second  infection.  Artificial  immunity  to  infection  can  also  be 
produced  in  various  ways:  by  inoculations  with  cultures  which  have 
been  kept  for  a  few  days  at  a  temperature  of  42°  to  43°  C.  and 
have  thus  lost  their  original  virulence,  or  by  inoculations  of  filtered 
cultures,  or  of  cultures  sterilized  by  heat.  For  the  production  of 


392  BACTERIA  PATHOGENIC  TO  MAN 

immunity  in  cattle  it  is  advised  to  use  a  dried  powder  of  the  muscles 
of  animals  which  have  succumbed  to  the  disease,  and  which  have  been 
subjected  to  a  suitable  temperature  to  ensure  attenuation  of  the  viru- 
lence of  the  spores  contained  therein.  Two  vaccines  are  prepared,  as 
in  anthrax — a  stronger  vaccine  by  exposing  a  portion  of  the  powder  to 
a  temperature  of  85°  to  90°  C.  for  six  hours,  and  a  weaker  vaccine  by 
exposing  it  for  the  same  time  to  a  temperature  of  100°  to  104°  C.  In- 
oculations are  made  with  this  attenuated  virus  into  the  end  of  the  tail — 
first  the  weaker  and  later  the  stronger.  These  give  rise  to  a  local  reac- 
tion of  moderate  intensity,  and  the  animal  is  subsequently  immune  from 
the  effects  of  the  most  virulent  material  and  from  the  disease.  Fourteen 
days  are  allowed  to  elapse  between  the  two  inoculations.  The  results 
obtained  from  this  method  of  preventive  inoculation  seem  to  have  been 
very  satisfactory.  According  to  the  statistics,  including  many  thou- 
sand cattle  treated,  the  mortality,  which  among  22,300  non-inocu- 
lated cattle  was  2.20  per  cent.,  has  been  reduced  to  0.16  per  cent,  in 
14,700  animals  inoculated.  When  danger  of  immediate  infection 
exists,  it  is  advisable  to  inject  some  antitoxin  with  the  vaccine.  This 
lessens  the  reaction  and  gives  immediate  immunity. 


CHAPTER  XXX. 

THE  CHOLERA  SPIRILLUM  (SPIRILLUM   CHOLERA  ASIATICS) 
AND  ALLIED  VARIETIES. 


Ix  INV')  Koch  separated  a  characteristically  curved  organism  from 
the  dejecta  and  intestines  of  cholera  patients  —  the  so-called  "comma 
bacillus."  This  he  declared  to  be  absent  from  the  stools  and  intes- 
tinal contents  of  healthy  persons,  and  of  persons  suffering  from  other 
affections.  The  organism  was  said  to  possess  certain  morpholog'cal 
and  biological  features  which  readily  distinguished  it  from  all  previously 
described  organisms.  It  was  absent  from  the  blood  and  viscera,  and 
was  found  only  in  the  intestines;  and  the  greater  the  number,  it  was  said, 
the  more  acute  the  attack.  Koch  also  demonstrated  an  invasion  of 
the  mucosa  and  its  glands  by  this  "comma  bacilli."  The  organisms  were 
found  in  the  stools  on  staining  the  mucous  flakes  or  the  fluid  with 
methylene  blue  or  fuchsin,  and  sometimes  alone;  by  means  of  cultiva- 

Fir,.  120  FIG.  121 


Contact  smear  of  colony  of  cholera  spirilla  Cholera  spirilla  preparation  from  gelatin-plate 

from  a  gar.    X  700  diameters.    (Dunham.)  culture  of  cholera.    X  800  diameters. 

tion  on  gelatin  they  were  readily  separated  from  the  stools.  During 
his  stay  n  India,  in  Ku'vpt,  and' at  Toulon,  Koch  had  examined  over 
one  hundred  cases,  and  other  investigators  confirmed  his  statements. 
Numerous  control  observations  made  upon  other  diarrhoeic  dejecta 
and  upon  normal  stools  were  negative;  the  comma  bacillus  was  found 
in  choleraic  material  only,  or  in  material  contaminated  by  cholera. 
Soon,  however,  other  observers  described  comma-shaped  organisms 
of  non-choleraic  origin.  Kinkier  and  Prior,  for  instance,  found  them 
in  the  diarrhu*al  >t<>nlsnf  cholera  nostras,  Deneke  in  cheese, Lewis  and 
Miller  in  saliva.  All  of  these  organisms,  however,  differed  in  many 
respects  from  Koch's  comma  bacillus,  and  it  has  since  been  proved 


394:  BACTERIA  PATHOGENIC  TO  MAN 

that  none  of  them  is  affected  by  the  specific  serum  of  animals  immu- 
nized to  cholera.  After  a  time,  therefore,  the  exclusive  association  of 
Koch's  vibrio  with  cholera  became  almost  generally  acknowledged  until 
now  it  is  regarded  by  bacteriologists  everywhere  to  be  the  specific  cause 
of  Asiatic  cholera.  Certain  sporadic  cases  of  cholera-like  disease,  how- 
ever, are  undoubtedly  due  to  other  organisms. 

Morphology. — Curved  rods  with  rounded  ends  which  do  not  lie  in 
the  same  plane,  from  0.8/*  to  2ju  in  length  and  about  QAp  in  breadth. 
The  curvature  of  the  rods  may  be  very  slight,  like  that  of  a  comma, 
or  distinctly  marked,  particularly  in  fresh  unstained  preparations  of 
full-grown  individuals,  presenting  the  appearance  of  a  half-circle.  By  the 
inverse  junction  of  two  vibrios  S-shaped  forms  are  produced.  Longer 
forms  are  rarely  seen  in  the  intestinal  discharges  or  from  cultures  grown 
on  solid  media,  but  in  fluids,  especially  when  grown  under  unfavorable 
conditions,  long,  spiral  filaments  may  develop.  The  spiral  forms  are 
best  studied  in  the  hanging  drop,  for  in  the  dried  and  stained  prep- 
arations the  spiral  character  of  the  long  filaments  is  often  obliterated. 
In  film  preparations  from  the  intestinal  contents  in  typical  cases  it 
will  be  found  that  the  organisms  are  present  in  enormous  numbers, 
and  often  in  almost  pure  culture  (Figs.  120  and  121).  In  old  cultures 
irregularly  clubbed  and  thickened  involution  forms  are  frequent,  and 
the  presence  in  the  organisms  of  small,  rounded,  highly  refractile  bodies 
is  often  noted. 

Staining. — The  cholera  spirillum  stains  with  the  aniline  colors  usually 
employed,  but  not  as  readily  as  many  other  bacteria;  a  dilute  aqueous 
solution  of  carbol-fuchsin  is  recommended  as  the  most  reliable  staining 
agent  with  the  application  of  a  few  minutes'  heat.  It  is  decolorized  by 
Gram's  method.  The  organisms  exhibit  one  long,  fine,  spiral  flagellum 
attached  to  one  end  of  the  rods,  or,  exceptionally,  to  both  ends.  (Cholera- 
like  spirilla  often  have  1,  2,  or  3  end  flagella.) 

Biology. — An  aerobic  (facultative  anaerobic),  liquefying,  very  motile 
spirillum.  Grows  readily  in  the  ordinary  culture  media,  best  at  37°  C., 
but  also  at  room  temperature  (22°  C.);  does  not  grow  at  a  temperature 
above  42°  or  below  8°  C.  and  does  not  form  spores. 

In  gelatin-plate  cultures  at  22°  C.  the  colonies  are  quite  character- 
istic; at  the  end  of  twenty-four  hours,  small,  round,  yellowish-white  to 
yellow  colonies  may  be  seen  in  the  depths  of  the  gelatin,  which  later 
grow  toward  the  surface  and  cause  liquefaction  of  the  medium,  the 
colonies  lying  at  the  bottom  of  the  holes  or  pocket  thus  formed.  The 
zone  of  liquefaction,  which  increases  rapidly,  at  first  remains  clear, 
then  becomes  cloudy,  mostly  gray,  as  the  result  of  the  growth  of  the 
colonies.  In  many  cases  after  a  time  concentric  rings,  increasing  from 
day  to  day,  appear  in  the  zone  of  liquefaction.  (See  Figs.  122  and  123.) 
Examined  under  a  low-power  lens,  at  the  end  of  sixteen  to  twenty- 
four  hours,  the  colonies  appear  as  small,  light-yellow,  round,  coarsely 
granular  disks,  with  a  more  or  less  irregular  outline.  In  many  cases 
at  this  stage  an  ill-defined  halo  is  seen  to  surround  the  granular  colony. 
As  the  colonies  become  older  the  granular  structure  increases,  until  a 


THE  CHOLERA  SPIRILLUM  AND  ALLIED  VARIETIES 


395 


sta^e  is  reached  when  the  surface  looks  as  if  it  were  covered  with  little 
fragments  of  broken  glass.     Liquefaction  continues  about  the  colonies, 

their  structure  appears  fissured   and  coarsely  granular  in  texture,  and 
occasionally  a  hair-like  border  is  formed  at  the  periphery  (Fig.  123). 


Fie.  1-" 


FIG.  J23 


Cholera  colonies  in  gelatin  ;  twenty-four  to  thirty- 
six  hours'  growth.     X  about  20  diameters. 


Cholera  colony  in  gelatin,    x  30  diameters. 
(Dunham.) 


Sometimes  the  colonies  may  be  retained  as  compact  masses  in  the  zone 
of  liquefaction,  and  then  they  are  dark  yellow  or  brown  in  color,  and 
forms  occur  which  are  absolutely  unlike  the  typical  cholera  colonies. 


FIG.  1-J4 


A  characteristic  series  of  cholera  cultures  in  gelatin ;  one,  two,  three,  four,  and  six  days'  growth. 

(Dunham.) 

In  (/rlafui-*firl:  cultures  the  growth  is  at  first  thread-like  and  unchar- 
acteristic. At  the  end  of  twenty-four  to  thirty-six  hours  a  small,  funnel- 
shaped  depression  appears  on*  the  surface  of  the  gelatin,  which  soon 
spreads  out  in  the  form  of  an  air  bubble  above,  while  below  this  is  a 


396  BACTERIA  PATHOGENIC  TO  MAN 

whitish,  viscid  mass.  Later,  the  funnel  increases  in  depth  and  diame- 
ter, and  at  the  end  of  from  four  to  six  days  may  reach  the  edge  of  the 
test-tube;  in  from  eight  to  fourteen  days  the  upper  two-thirds  of  the 
gelatin  is  completely  liquefied.  (See  Fig.  124.)  Freshly  isolated  cholera 
vibrios  liquefy  gelatin  more  rapidly  than  old  laboratory  cultures;  a 
certain  variation,  under  some  circumstances,  in  the  characteristic  lique- 
faction of  the  gelatin  even  in  fresh  cultures,  should  be  borne  in  mind 
in  making  a  diagnosis.  Such  variations  in  cultural  peculiarities  occur 
also  with  other  bacteria. 

Upon  the  surface  of  agar  the  comma  bacillus  develops  a  moist,  shin- 
ing, grayish-yellow  layer.  In  agar-plate  cultures,  for  diagnostic  pur- 
poses, the  growth  of  separated  colonies  is  of  great  importance.  The 
nutrient  agar  after  pouring  in  the  plates  and  solidifying  should  be 
slightly  dried  on  the  surface  by  putting  the  uncovered  plate  face  down- 
ward on  the  shelf  of  the  incubator  at  37°  C.  for  thirty  minutes,  or  at 
60°  C.  for  five  minutes.  The  cholera  colonies  develop  fairly  charac- 
teristically, being  more  transparent  than  those  of  most  other  bacteria 
except  the  cholera-like  vibrios.  Blood  serum  is  rapidly  liquefied  at 
the  temperature  of  the  incubator.  On  potato  at  incubator  temperature 
a  moist  growth  of  a  dirty-brown  color  occurs.  Milk  is  not  coagulated. 
In  bouillon  the  growth  is  rapid  arid  abundant;  in  the  incubator  at  the 
end  of  ten  to  sixteen  hours  the  liquid  is  diffusely  clouded,  and  on  the 
surface  a  wrinkled  membranous  layer  is  often  formed.  In  general 
the  spirillum  grows  in  any  liquid  containing  a  small  quantity  of  organic 
matter  and  having  a  slightly  alkaline  reaction.  An  acid  reaction  of 
the  culture  medium  prevents  its  development,  as  a  rule;  but  it  has  the 
power  of  gradually  accommodating  itself  to  the  presence  of  vegetable 
acids.  Abundant  development  occurs  in  bouillon  which  has  been 
diluted  with  eight  to  ten  parts  of  water  and  in  simple  peptone  solution. 

The  comma  bacillus  belongs  to  the  class  of  aerobic  organisms,  inas- 
much as  it  grows  readily  only  in  the  presence  of  oxygen,  and  that  it 
develops  active  motility  only  when  a  certain  amount  of  oxygen  is 
present.  It  does  not  grow  in  the  total  absence  of  oxygen,  but  a  small 
quantity  of  oxygen  is  all  that  is  required  for  its  development,  as  in  the 
intestines.  This  need  of  oxygen  tends  to  send  the  spirilla  to  the  surface 
of  fluid  culture  media. 

CHOLERA-RED  REACTION. — When  a  small  quantity  of  chemically  pure 
sulphuric  acid  is  added  to  a  twenty-four-hour  bouillon  culture  of  the 
cholera  bacillus  containing  peptone  a  reddish-violet  color  is  produced. 
Brieger  separated  the  pigment  formed  in  this  reaction — the  so-called 
cholera-red — and  showed  that  it  was  indol,  and  that  the  reaction  was 
nothing  more  than  the  well-known  indol  reaction.  Salkowski  and  Petri 
then  demonstrated  that  the  cholera  bacilli  produced  in  thin  bouillon 
cultures,  along  with  indol,  nitrites  by  reducing  the  nitrates  contained 
in  small  quantities  in  the  culture  media.  They  showed  that  it  is  the 
nitric  acid,  liberated  by  the  addition  of  sulphuric  acid  to  the  culture, 
which  gives  rise  to  the  indol,  the  red  body  upon  which  the  cholera 
reaction  depends.  For  a  long  time  it  was  belie ved  that  this  nitroso- 


777 /•;  CHOLERA   SPIiUU.f.M   AM)  M.IJl-D   VARIETIES  397 

indol  reaction  was  peculiar  to  tlic  cholera  bacillus,  and  great  weight  was 
])laccd  on  it  as  a  diagnostic  test.  It  lias  since  been  shown,  however,  tl  at 
there  arc  a  number  of  other  vibrios  which,  under  similar  condition 
the  cholera  vibrio,  give  the  same  red  reaction.  The  reaction  i>,  neverthe- 
less, a  constant  and  characteristic  peculiarity  of  this  spirillum,  and  is  of 
unquestionable  value.  It  is  even  more  valuable  as  a  negative  than  as  a 
positive  test,  as  the  absence  of  the  reaction  enables  one  to  say  of  a  sus- 
pected organism  that  it  is  not  the  cholera  spirillum.  There  are,  how- 
ever, certain  precautions  to  be  observed  in  its  use.  It  has  been  shown 
that  the  reaction  may  be  absent,  for  instance,  when  the  culture  contain.-* 
either  too  much  or  too  little  nitrate.  It  is,  therefore,  advisable  not  to 
employ  a  bouillon  culture  the  composition  of  which  is  uncertain,  but 
a  distinctly  alkaline  solution  of  peptone,  containing  1  per  cent,  pure 
peptone  and  0.5  per  cent,  of  pure  chloride  of  sodium  (Dunham's  solu- 
tion). With  such  a  solution  constant  results  can  be  obtained. 

DEVELOPMENT  OUTSIDE  OF  THE  BODY. — It  has  been  shown  by 
experiment  that  cholera  spirilla  multiply  to  some  extent  in  sterilized 
river-water  or  well-water,  and  preserve  their  vitality  in  such  water  for 
several  weeks  or  even  months.  Koch  demonstrated  the  presence  of 
this  spirillum  in  the  foul  water  of  a  tank  in  India  which  was  used  by 
the  natives  for  drinking  purposes.  In  his  early  investigations  he  found 
that  rapid  multiplication  may  occur  upon  the  surface  of  moist  linen. 

RESISTANCE  AND  VITALITY. — If  a  culture  be  spread  on  a  cover-glass 
and  exposed  to  the  action  of  the  air  at  room  temperature  the  bacilli 
will  be  dead  at  the  end  of  two  or  three  hours,  unless  the  layer  of  culture 
is  very  thick,  in  which  case  it  may  take  twenty-four  hours  or  more  to 
kill  all  the  bacilli.  This  indicates  that  infection  is  not  produced  by  means 
of  dust  or  other  dried  objects  contaminated  with  cholera  bacilli.  The 
transmission  of  these  organisms  through  the  air,  therefore,  can  only 
take  place  for  short  distances,  as  by  a  spray  of  infectious  liquids  by 
mechanical  means — as,  for  instance,  the  breaking  of  waves  in  a  har- 
bor, on  water-wheels,  etc.,  or  in  moist  wash  of  cholera  patients. 

The  cholera  bacillus  is  also  injuriously  affected  by  the  abundant 
growth  of  saprophytic  bacteria.  It  is  true  that  when  associated  with 
other  bacteria,  if  present  in  large  numbers,  and  if  the  conditions  for 
their  development  are  particularly  favorable,  the  cholera  bacillus  may 
at  first  gain  the  upper  hand,  as  in  the  moist  linen  of  cholera  patients, 
or  in  soil  impregnated  with  cholera  dejecta;  but  later,  after  two  or 
three  days,  even  in  such  cases,  the  bacilli  die  off  and  other  bacteria  grad- 
ually take  their  place.  Thus,  Koch  found  that  the  fluid  contents  of 
privies  twenty-four  hours  after  the  introduction  of  comma  bacilli  no 
longer  contained  the  living  organisms;  in  impure  river-water  they  were 
not  demonstrable  for  more  than  six  to  seven  days,  as  a  rule.  In  the 
dejecta  of  cholera  patients  they  were  found  usually  only  for  a  few  days 
Cone  to  three  days),  though  rarely  they  have  been  observed  for  twenty 
to  thirty  days,  and  on  one  occasion  for  one  hundred  and  twenty  days. 
In  unsterili/ed  water  they  may  also  retain  their  vitality  for  a  relatively 
longtime;  thus,  in  stagnant  well-water  they  have  been  found  for  eighteen 


398  BACTERIA  PATHOGENIC  TO  MAN 

days,  and  in  an  aquarium  containing  plants  and  fishes,  the  water  of 
which  was  inoculated  with  cholera  germs,  they  were  isolated  several 
months  later  from  the  mud  at  the  bottom.  In  running  river-water, 
however,  they  have  not  been  observed  for  over  six  to  eight  days.  For 
the  cholera  organisms  the  conditions  favorable  to  growth  are  a  warm 
temperature,  moisture,  a  good  supply  of  oxygen,  and  a  considerable 
proportion  of  organic  material.  These  conditions  are  fully  met  with 
outside  the  body  in  but  very  few  localities. 

The  comma  bacillus  has  the  average  resistance  of  spore-free  bac- 
teria, and  is  killed  by  exposure  to  moist  heat  at  60°  C.  in  ten  minutes, 
at  95°  to  100°  C.  in  one  minute.  The  bacilli  have  been  found  alive 
kept  for  a  few  days  in  ice,  but  ice  which  has  been  preserved  for  several 
weeks  does  not  contain  living  bacilli. 

Chemical  disinfectants  readily  destroy  the  vitality  of  cholera  vibrios. 
For  disinfection  on  a  small  scale,  as  for  washing  the  hands  when  con- 
taminated with  cholera  infection,  a  0.1  per  cent,  solution  of  bichloride 
of  mercury  or  a  2  to  3  per  cent,  solution  of  carbolic  acid  may  be  used. 
For  disinfection  on  a  large  scale,  as  for  the  disinfection  of  cholera  stools, 
strongly  alkaline  milk  of  lime  is  an  excellent  agent.  The  wash  of  cholera 
patients,  contaminated  furniture,  floors,  etc.,  may  be  disinfected  by  a 
solution  of  5  per  cent,  carbolic  acid  and  soap  water. 

The  Spread  of  Cholera. — Cholera  is  practically  always  transmitted 
by  means  of  water  or  food  contaminated  by  the  spirilla,  and  there  is 
no  doubt  that  the  contamination  is  in  most  all  cases  through  the  direct 
soiling  of  the  water  by  the  feces  of  cholera  patients.  Flies  which  have 
fed  or  lighted  on  the  discharges  of  cholera  patients  or  on  things  con- 
taminated by  them  have  been  found  to  carry  the  organisms  not  only 
on  their  feet,  but  also  in  their  bodies  for  at  least  twenty-four  hours. 
Food  contaminated  by  flies  is  therefore  a  possible  source  of  infection. 

Pathogenesis. — None  of  the  lower  animals  is  naturally  subject  to 
cholera,  nor  has  any  contracted  the  disease  as  the  result  of  the  ingestion 
of  food  contaminated  with  choleraic  excreta  or  from  the  inoculations 
of  pure  cultures  of  the  spirillum,  either  subcutaneously  or  by  the  mouth. 
It  has  been  shown  that  the  comma  bacillus  is  extremely  sensitive  to 
the  action  of  acids,  and  is  quickly  destroyed  by  the  acid  secretions  of 
the  stomach  of  man  or  the  lower  animals,  when  these  secretions  are 
normally  produced.  Nikati  and  Rietsch  produced  a  choleraic  condi- 
tion in  a  considerable  percentage  of  dogs,  where  the  virulent  cultures 
were  injected  directly  into  the  duodenum.  Koch  sought  to  produce 
infection  in  guinea-pigs  per  vias  naturales  by  first  neutralizing  the 
contents  of  the  stomach  with  a  solution  of  carbonate  of  soda — 5  c.c. 
of  a  5  per  cent,  solution  injected  into  the  stomach  through  a  pharyngeal 
catheter — and  then  after  a  while  administered  through  a  similar  catheter 
10  c.c.  of  a  liquid  into  which  had  been  put  one  or  two  drops  of  a  bouillon 
culture  of  the  comma  bacillus.  The  animal  then  receives  a  dose  of  1  c.c. 
of  tincture  of  opium  per  200  grams  of  body-weight,  introduced  into 
the  abdominal  cavity,  for  the  purpose  of  controlling  the  peristaltic  move- 
ments. As  a  result  of  this  treatment  the  animals  are  completely  nar- 


THE  CHOLERA  SPIHILIA'M  AM)  ALLIED  VARIETIES          399 

for  about  half  an  hour,  Imt  recover  from  it  without  showing 
any  ill  effects.  On  the  evening  of  the  same  or  the  following  day  the 
animal  shows  an  indisposition  to  eat  and  other  signs  of  weakness,  its 
posterior  extremities  become  weak  and  apparently  paralyzed,  and,  as 
a  rule,  death  occurs  within  forty-eight  hours  with  the  symptoms  of 
collapse  and  fall  of  temperature.  At  the  autopsy  the  small  intestine 
is  found  to  be  congested  and  filled  with  a  watery  fluid,  containing  the 
spirillum  in  great  numbers.  Koch  experimented  in  this  way  on  about 
one  hundred  guinea-pigs.  These  results,  however,  are  somewhat 
weakened  by  the  fact  that  experiments  made  with  some  other  bacteria 
morphologically  similar  to  the  comma  bacillus  of  Koch,  but  specific- 
ally different,  occasionally  produced  death  when  introduced  in  the 
same  way  into  the  small  intestines  of  guinea-pigs.  Metchnikoff  dis- 
covered that  young  rabbits  shortly  after  birth  could  be  infected  by 
simply  infecting  the  teats  of  the  mother  so  that  they  received  infection 
along  with  the  milk. 

There  are  several  cases  on  record  which  furnish  the  most  satisfactory 
evidence  that  the  cholera  spirillum  is  able  to  produce  the  disease  in 
man.  In  1884  a  student  in  Koch's  laboratory  in  Berlin,  who  was 
taking  a  course  on  cholera,  became  ill  with  a  severe  attack  of  cholera. 
At  that  time  there  was  no  cholera  in  Germany,  and  the  infection  could 
not  have  been  produced  in  any  other  way  than  through  the  cholera 
cultures  which  were  being  used  for  the  instruction  of  students.  In 
1892  Pettenkofer  and  Emmerich  experimented  on  themselves  by 
swallowing  small  quantities  of  fresh  cholera  cultures  obtained  from 
Hamburg.  Pettenkofer  was  affected  with  a  mild  attack  of  cholerine 
or  severe  diarrhoea,  from  which  he  recovered  in  a  few  days  without 
any  serious  effects;  but  Emmerich  became  very  ill.  On  the  night  fol- 
lowing the  infection  he  was  attacked  by  frequent  evacuations  of  the 
characteristic  rice-water  type,  cramps,  tympanites,  and  great  prostra- 
tion. His  voice  became  hoarse,  and  the  secretion  of  urine  was  some- 
what diminished,  this  condition  lasting  for  several  days.  In  both  cases 
the  cholera  spirillum  was  obtained  in  pure  culture  from  the  dejecta. 
Another  instance  is  reported  by  Metchnikoff,  in  Paris,  of  a  man  who 
became  infected  experimentally.  In  this  case  the  algid  stage  of  cholera 
was  produced,  with  complete  suppression  of  urine,  cramps  in  the  legs, 
contraction  of  the  extremities,  and  collapse,  the  man's  life  being  saved 
only  with  difficulty.  Finally,  there  is  the  case  of  Dr.  Oergel,  of  Ham- 
burg, who  accidentally,  while  experimenting  on  a  guinea-pig,  had 
some  of  the  infected  peritoneal  fluid  to  squirt  into  his  mouth.  He  was 
taken  ill  and  died  a  few  days  afterward  of  typical  cholera,  though  at 
the  time  of  his  death  there  was  no  cholera  in  the  city.  These  accidents 
and  experiments  would  certainly  seem  to  prove  conclusively  the  capa- 
bility of  pure  cholera  cultures  which  have  retained  their  virulence  of 
producing  the  disease. 

Lesions  in  Man. — Cholera  in  man  is  an  infective  process  of  the  epithe- 
lium of  the  intestine,  in  which  the  spirilla  clinging  to  and  between  the 
epithelial  cells  produce  a  partial  or  entire  necrosis  and  final  destruction 


400  BACTERIA  PATHOGENIC  TO  MAN 

of  the  epithelial  covering,  which  thus  renders  possible  the  absorption 
of  the  cholera  toxin  formed  by  the  growth  of  the  spirilla.  The  larger 
the  surface  of  the  mucous  membrane  infected  and  the  more  luxuriant  the 
development  of  bacilli  and  the  production  of  toxin  the  more  pronounced 
will  be  the  poisoning,  ending  fatally  in  a  toxic  paralysis  of  the  circu- 
latory and  thermic  centres.  On  the  other  hand,  however,  there  may 
be  cases  where,  in  spite  of  the  large  number  of  cholera  bacilli  present 
in  the  dejecta,  severe  symptoms  of  intoxication  may  be  absent.  In 
such  cases  the  destruction  of  epithelium  is  not  produced  or  is  so  slight 
that  the  toxic  substance  absorbed  is  not  in  sufficient  concentration  to 
give  rise  to  the  algid  stage  of  the  disease,  or  for  some  reason  the  spirilla 
do  not  produce  toxin  to  any  extent.  In  no  stage  of  the  disease  are 
living  cholera  spirilla  found  in  the  organs  of  the  body  or  in  the  secre- 
tions. 

Distribution  in  the  Body. — The  cholera  spirilla  are  found  only  in  the 
intestines  and  are  believed  never  to  be  present  in  the  blood  or  internal 
organs.  The  lower  half  of  the  small  intestine  is  most  affected,  a  large 
part  of  its  surface  epithelium  becoming  shed.  The  flakes  floating  in 
the  rice-water  discharges  consist  mostly  of  masses  of  epithelial  cells 
and  mucus,  among  which  are  numerous  spirilla.  The  spirilla  also 
penetrate  the  follicles  of  Lieberkiihn,  and  may  be  seen  lying  between 
the  basement-membrane  and  the  epithelial  lining,  which  become 
loosened  by  their  action.  They  are  rarely  found  in  the  connective 
tissue  beneath,  and  never  penetrate  deeply.  In  more  chronic  cases 
other  micro-organisms  play  a  greater  part  and  deeper  lesions  of  the 
intestines  may  occur. 

Communicability. — From  this  fact  and  other  known  properties  of  the 
cholera  spirillum,  which  have  already  been  referred  to,  several  im- 
portant deductions  may  be  made  with  regard  to  the  mode  of  transmis- 
sion of  cholera  infection.  In  the  first  place  the  bacilli  evidently  leave 
the  bodies  of  cholera  patients,  chiefly  in  the  dejections  during  the 
early  part  of  the  disease  (they  have  usually  disappeared  after  the 
fourth  to  the  fourteenth  day),  and  only  these  dejections,  therefore, 
and  objects  contaminated  by  them,  such  as  bed  and  body  linen, 
floors,  vaults,  soil,  well-water  and  river-water,  etc.,  can  be  regarded 
as  possible  sources  of  infection.  There  is  a  special  limitation  even 
in  these  sources  of  infection,  owing  to  the  fact  that  this  spirillum  is 
so  easily  destroyed  by  desiccation  and  crowded  out  by  saprophytic 
organisms.  Thus,  as  a  rule,  only  fresh  dejections  and  freshly  con- 
taminated objects  are  liable  to  convey  infection;  after  they  have 
become  completely  dry  there  is  little  danger.  Further,  we  must  con- 
clude from  the  distribution  of  the  cholera  bacillus  in  the  body  and  from 
experiments  upon  animals  that  the  commonest  mode  of  infection  is  by 
way  of  the  mouth,  and  chiefly  by  means  of  water  used  for  drinking  pur- 
poses, for  the  preparation  of  food,  etc.  In  recent  times  cholera  spirilla 
have  been  found  not  infrequently  in  water  (wells,  water-mains,  rivers, 
harbors,  and  canals)  which  has  become  contaminated  by  the  .dejec- 
tions of  cholera  patients. 


THE  CHOLERA  SPIRILLUM  AND  ALLIED  VARIETIES         401 

As  in  like  other  infectious  diseases,  not  everyone  who  is  exposed  to 
infection  is  attacked  by  cholera.  The  bacilli  have  been  found  during 
cholera  epidemics  in  the  dejections  of  healthy  individuals  without  any 
pathological  symptoms.  Abel  and  Claussen  for  example,  in  14  out 
of  17  persons  belonging  to  the  families  .of  7  cholera  patients,  found 
cholera  vibrios,  in  some  of  them  for  a  period  of  fourteen  days.  In 
Hamburg  there  were  28  such  cases  of  healthy  choleraic  individuals 
with  absolutely  normal  stools.  It  is  evident,  therefore,  that  an  individual 
susceptibility  is  requisite  to  produce  the  disease.  In  the  normal  healthy 
stomach  the  hydrochloric  acid  of  the  gastric  secretions  may  destroy 
the  spirilla;  and,  finally,  the  normal  vital  resistance  of  the  tissue  cells 
to  the  action  of  the  cholera  poison  may  be  taken  into  consideration. 
According  to  the  greater  or  less  power  of  this  vital  resistance  of  the  body 
the  same  infectious  matter  may  give  rise  to  no  disturbance  whatever, 
a  slight  diarrhoea,  or  it  may  lead  to  serious  results.  Furthermore,  it 
may  be  accepted  as  an  established  fact,  that  recovery  from  one  attack 
of  cholera  produces  personal  immunity  to  a  second  attack  for  a  con- 
siderable length  of  time.  This  does  not  appear  to  depend  upon  the 
severity  of  the  attack;  for  cases  are  recorded  of  persons  who  were  appar- 
ently not  sick  at  all,  and  yet  in  whom  an  acquired  immunity  was  pro- 
duced. How  long  this  immunity  lasts  is  not  positively  known,  but 
probably  for  a  month  or  more,  so  that  the  same  person  is  not  likely 
to  be  taken  ill  again  with  cholera  during  an  epidemic. 

On  the  other  hand,  we  may  take  it  for  granted  that  susceptibility  to 
cholera  may  be  acquired  or  increased.  For  instance,  there  is  no  doubt 
that  gastric  and  intestinal  disorders  produced  by  overeating,  etc.,  may 
act  as  contributing  causes  to  the  disease.  Other  predisposing  causes 
are  general  debility  from  poverty,  hunger,  disease,  etc. 

Cholera  Toxins. — Koch  was  the  first  to  assume,  as  the  result  of  his 
investigations,  that  the  severe  symptoms  of  the  algid  stage  of  cholera 
were  due  to  the  effects  of  a  toxin  produced  by  the  growth  of  the  comma 
bacillus  in  the  intestines. 

In  1892  Pfeiffer  published  an  account  of  his  elaborate  researches 
relating  to  the  cholera  poison.  He  found  that  recent  aerobic  cultures 
of  the  cholera  spirillum  contain  a  specific  toxic  substance  which  is  fatal 
to  guinea-pigs  in  extremely  small  doses.  There  is  extreme  collapse, 
with  subnormal  temperature.  This  substance  stands  in  close  relation 
with  the  bacterial  cells,  and  is  perhaps  an  integral  part  of  them.  The 
filtrate  of  a  recent  cholera  culture  contains  usually  only  moderate 
amounts  of  toxic  substances.  The  spirilla  may  be  killed  by  chloroform, 
thymol,  or  by  desiccation,  without  apparent  injury  to  the  toxic  power 
of  this  substance,  but  subjected  to  60°  C.  some  of  the  toxins  are  destroyed. 
Metchnikoff,  Roux  and  others  have  shown  that  living,  highly  virulent 
cultures  produce  at  times  highly  poisonous  toxins,  the  0.2  c.c.  of  filtrate 
of  a  three  to  four  day  culture  killing  100  grams  of  guinea-pig.  The 
living  culture  in  2  to  4  c.c.  of  nutrient  bouillon  contained  in  collodion  sacs, 
when  placed  in  the  peritoneal  cavity  of  guinea-pigs,  produced  symptoms 
of  poisoning  and  death  in  a  few  days.  Sacs  containing  the  dead  vibrios 

26 


402  BACTERIA  PATHOGENIC  TO  MAN 

produced  little  effect.  There  appears  to  be,  therefore,  considerable 
difference  between  the  intracellular  and  the  soluble  extracellular 
toxins. 

Cholera  Immunity. — Koch  found  in  his  animal  experiments  that 
recovery  from  an  intraperitoneal  infection  with  small  doses  of  living 
cholera  vibrios  produced  a  certain  immunity  against  larger  doses, 
though  the  animals  inoculated  were  not  very  much  more  resistant  to 
the  cholera  poison  than  they  were  originally.  In  1892  Lazarus  observed 
that  the  blood  serum  of  persons  who  had  recently  recovered  from  an 
attack  of  cholera  possessed  the  power  of  preventing  the  development 
in  guinea-pigs  of  cholera  bacilli,  which  in  these  animals  are  rapidly 
fatal  when  injected  intraperitoneally,  while  the  serum  of  healthy  indi- 
viduals had  no  such  effect.  This  specific  change  in  the  blood  is  observed 
to  take  place  from  eight  to  ten  days  after  the  termination  of  an  attack 
of  cholera,  and  reaches  its  maximum  during  the  fourth  week  of  con- 
valescence, after  which  it  declines  rapidly  and  disappears  entirely  in 
about  two  or  three  months.  Similar  antitoxic  or  bactericidal  sub- 
stances develop  in  the  serum  of  guinea-pigs,  rabbits,  and  goats,  when 
these  animals  are  immunized  artificially  against  cholera  by  subcuta- 
neous or  intraperitoneal  injections  of  living  or  dead  cultures.  These 
specific  substances  present  in  the  blood  of  cholera-immune  men  and 
animals  act  only  upon  organisms  similar  to  those  with  which  they  were 
infected;  but,  as  Pfeiffer  showed,  this  specific  relation,  which  is  found 
to  exist  between  the  antibacterial  and  protective  substances  produced 
during  immunization  and  the  bacteria  employed  to  immunize  the 
animals,  is  not  confined  to  cholera.  The  discovery,  moreover,  of  this 
specific  reaction  of  the  blood  serum  of  immunized  man  and  animals 
when  brought  in  contact  with  the  spirilla,  has  given  us  an  apparently 
reliable  means  of  distinguishing  the  cholera  from  all  other  vibrios,  and 
the  disease  cholera  from  other  similar  affections,  both  of  which 
have  proved  to  be  of  great  value,  particularly  in  obscure  or  doubtful 
cases,  in  which  heretofore  the  only  method  of  differential  diagnosis 
available — viz.,  by  cultural  tests — was  often  unsatisfactory. 

Anticholera  Inoculations. — Within  the  last  few  years  Haffkine,  in 
India,  has  succeeded  in  producing  an  artificial  immunity  against  cholera 
infection  by  means  of  subcutaneous  injections  of  cholera  cultures.  Two 
or  three  injections  are  necessary  to  give  the  greatest  amount  of  protec- 
tion. Animals  treated  by  this  method  are  refractory  to  intraperitoneal 
inoculations,  but  not  to  intestinal  injections  or  feeding  by  Koch's  method. 
In  the  intestines  the  bacteria  seem  to  be  outside  the  influence  of  the 
bactericidal  properties  of  the  blood,  and  the  absorption  of  toxins  is  too 
great  to  be  neutralized  by  the  small  amount  of  antitoxin.  In  over 
200,000  persons  whom  he  has  inoculated  the  results  obtained  would 
undoubtedly  seem  to  show  a  distinct  protective  influence  in  the  pre- 
ventive inoculations. 

Agglutinins. — Five  to  ten  days  after  infection   (natural  or  experi- 
mental) agglutinins  appear  in  the  blood  of  man  or  animal.     These  \ 
are  at  least  in  part  specific.    Their  presence  in  the  blood  is  of  diagnostic 


THE  CHOLERA  SPIRILLUM  AND  ALLIED  VARIETIES         403 

importance.  When  present  in  great  amount  such  agglutinins  can  be 
used  for  identifying  doubtful  spirilla. 

Variations  of  the  Cholera  Spirillum. — From  the  great  majority  of 
all  cases  of  epidemic  cholera  examined,  cholera  spirilla  agreeing  in 
all  essential  characteristics  have  been  obtained,  usually  in  great 
numbers  and  often  in  almost  pure  culture.  In  their  agglutination 
with  a  specific  serum  they  are  also  alike.  Some  cultures  agglutinate 
with  more  difficulty  than  others,  so  that  the  same  serum  may  agglutinate 
different  cultures  in  dilutions  varying  from  1 : 1000  up  to  1 : 10,000. 
Such  a  serum  would  not  agglutinate  cholera-like  spirilla  above  a  1 : 50 
dilution.  Especially  among  isolated  cases  of  cholera-like  diseases 
spirilla  are  met  with  which  do  not  agree  in  agglutination  charac- 
teristics. 

Biological  Diagnosis  of  the  Cholera  Vibrio.  Plan  of  Procedure. — A. 
Dejecta  (fluid)  or  intestinal  contents  of  a  cholera  patient  or  cholera 
suspect. 

1.  Use  one  drop  (one  platinum  loop)  for  gelatin-plate  cultures,  mak- 
ing two  dilutions.    Do  this  in  duplicate  or  triplicate.    Cultivate  at  22°  C. 

2.  Inoculate  a  couple  of  bouillon  tubes  and  a  couple  of  Dunham's 
1  per  cent,  peptone  solution  with  one  drop  each,  and  place  them  in  the 
incubator  (37°  to  38°  C.)  for  six  to  eight  hours. 

3.  Examine  a  drop  of  the  dejecta  in  the  hanging  drop. 

4.  Examine  a  drop  of  the  dejecta  in  stained  cover-glass  preparation.1 

5.  Make  gelatin  plates  from  one  drop  taken  from  the  surface  of  each 
of  the  bouillon  and  peptone  solution  tubes  and  cultivate  at  22°  C. 

6.  As  soon  as  the  plates   (see  1  and  5)  are  sufficiently  developed 
(thirty-six  to  forty-eight   hours)   fish  the  suspected   cholera  colonies 
and  use  the  material  for  the  following  procedures: 

7.  Inoculate  six  or  eight  peptone  tubes  (1  per  cent,  peptone  and  0.5 
per  cent.  NaCl  in  distilled  water)  and  place  them  at  once  in  the  incu- 
bator.    Note  the  time. 

8.  Examine  hanging  drop  for  form,  size,  and  motility  (and  arrange- 
ment). 

9.  Make  stained  cover-glass  preparations  and  examine. 

10.  Then  try  indol  reaction  with  the  same  tubes. 

11.  While  these  tubes  are  incubating  use  material  from  the  suspected 
colonies  on  the  plates  (1  and  5)  for  hanging-drop  cultures. 

12.  Meanwhile  make  stained  cover-glass   preparations  from  other 
colonies  of  suspected  cholera  on  the  plates  (1  and  5). 

13.  Make  gelatin-tube  cultures  from  colonies  on  plates  (1  and  5). 

14.  Make  gelatin-tube  cultures  daily  for  five  or  six  days,  to  study 
shape  of  growth  along  the  line  of  puncture  to  preserve  the  culture. 

1  These  direct  microscopic  examinations  of  the  intestinal  contents  are,  as  a  rule,  very  unsatis- 
factory, at  least  in  those  in  which  the  symptoms  are  not  marked.  In  a  few  the  spirals  will  make  up 
from  .50  to  100  per  cent,  of  the  bacteria  present.  In  most  of  the  cases  during  the  last  epidemic  in 
New  York  Dunham  found  abundance  of  columnar  epithelium  from  the  intestinal  mucous  mem- 
brane, numerous  straight,  thick  bacilli,  and  only  a  few  curved  bacilli  or  segments  of  spirals— too  few 
to  identify.  Plate  cultures  from  these  showed  from  20  to  80  per  cent,  of  all  the  colonies  developing 
to  be  cholera  spirilla. 


404  BACTERIA  PATHOGENIC  TO  MAN 

B.  Suspected  water. 

Add  to  500  c.c.  or  1  litre  of  the  water  to  be  examined  in  a  flask  half- 
full  enough  peptone-salt  solution  (20  per  cent,  peptone  and  10  per  cent. 
NaCl)  to  make  a  1  per  cent,  solution  of  peptone.  Then  proceed  as 
in  A. 

SPECIFIC  SERUM  REACTIONS. — All  authors  now  agree  that  the  differ- 
entiation of  the  cholera  vibrio  from  other  similar  vibrios  cannot  always 
be  made  by  the  cultural  method,  nor  is  the  usual  inoculation  of  animals 
sufficient.  For  this  purpose  serum  is  employed  either  by  making  intra- 
peritoneal  injections  of  a  surely  fatal  dose  of  the  suspected  spirillum 
along  with  the  serum  of  animals  immunized  to  undoubted  cholera 
cultures,  or  to  note  whether  specific  protection  is  afforded,  or  the 
Gruber-Widal  test  is  carried  out  in  such  a  way  as  to  determine  if  spe- 
cific agglutination  of  the  spirilla  occurs. 

Spirilla  More  or  Less  Allied  to  the  Cholera  Spirillum. 

The  examinations  of  the  stools  of  persons  suffering  from  cholera 
have  revealed,  in  a  small  percentage  of  cases,  spirilla  resembling  either 
very  closely  or  having  a  fair  degree  of  similarity  to  the  true  cholera 
organisms.  Further,  in  a  small  percentage  of  cases  having  choleraic 
symptoms  no  true  cholera  vibrios  have  been  found,  but  instead  other 
spirilla  resembling  them  more  or  less  closely. 

These  may  differ  in  having  two  or  more  end  flagella,  in  size,  in  pro- 
duction of  nitrites,  etc.,  or  they  may  be  identical  in  the  tests  commonly 
employed.  They  all  differ  in  the  specific  agglutination  and  bacterio- 
lytic  tests  from  the  cholera  spirilla  and  among  themselves. 

In  a  recent  epidemic  in  Egypt,  Gottschlich  obtained  from  sixteen  cases 
spirilla  differing  from  the  true  spirilla,  and  found  every  one  distinct 
in  some  characteristic  from  all  others.  Some  were  pathogenic  for 
pigeons,  through  inoculation  of  a  small  quantity  into  the  breast  muscle ; 
others  were  atypical  in  their  development  in  nutrient  gelatin.  None 
of  these  micro-organisms  injected  into  animals  induced  production  of 
agglutinins  for  the  true  cholera  spirilla. 

Kolle  and  Gottschlich  consider  these  various  spirilla  found  by  them 
in  Egypt  as  well  as  others  found  by  different  investigators  in  India, 
Germany,  and  elsewhere  to  be  saprophytes.  It  is  more  probable,  in 
the  writer's  opinion,  that  some  of  them  must  be  considered  as  bearing 
a  part  in  exciting  a  cholera-like  disease,  but  that  they  are  not  very 
pathogenic  and  require  very  favorable  conditions  before  they  can  exert 
their  action. 

Some  special  varieties  of  spirilla  resembling  those  of  cholera  have 
received  especial  attention  on  account  of  having  been  obtained  before 
it  was  known  that  so  many  cholera-like  vibrios  existed.  The  vibrio 
Berolinensis,  cultivated  by  Neisser  from  Berlin  sewage-water;  the 
vibrio  Danubicus,  cultivated  by  Hausser  from  canal-water,  and  the 
vibrio  of  Massowah,  cultivated  by  Pasquale  from  a  case  during  an 
epidemic  of  cholera,  all  are  negative  to  the  specific  serum  reactions, 


THE  CHOLERA  SPIRILLUM  AND  ALLIED  VARIETIES         405 

and  differ  in  the  number  of  terminal  flagella  or  in  other  characteristics. 
Cunningham  found  a  number  of  such  spirilla  in  cases  of  apparently 
true  cholera  in  India.  Some  of  these  may  have  been  true  cholera 
spirilla  and  others  may  have  had  some  relationship  to  the  disease  in 
the  person  from  which  they  were  derived. 

Spirillum  of  Finkler  and  Prior. 

Because  of  their  prominence  in  literature  and  their  frequent  use  in 
teaching,  the  spirillum  of  Finkler  and  Prior,  that  of  Metchnikoff,  and 
that  of  Deneke  are  of  considerable  interest. 

Finkler  and  Prior,  in  1884,  obtained  from  the  feces  of  patients  with 
cholera  nostras,  after  allowing  the  dejecta  to  stand  for  some  days,  a 
spirillum  which  is  of  interest  mainly  because  it  simulates  the  comma 
bacillus  of  Koch,  but  differs  from  it  in  several  cultural  peculiarities. 


FIG. 125 


Spirillum  or  Finkler  and  Prior,    x  1100  diameters. 

Morphology. — Somewhat  longer  and  thicker  than  the  spirillum  of 
Asiatic  cholera  and  not  so  uniform  in  diameter,  the  central  portion 
being  usually  wider  than  the  pointed  ends. 

Biology. — An  aerobic  and  facultative  anaerobic,  liquefying  spirillum. 
Does  not  form  spores.  Upon  gelatin  plates  small,  white,  punctiform 
colonies  are  developed  at  the  end  of  twenty-four  hours.  These  are 
round,  but  less  coarsely  granular,  darker  in  color,  and  with  a  more 
sharply  defined  border  than  the  comma  bacillus.  Liquefaction  of 
the  gelatin  around  these  colonies  progresses  rapidly,  and  at  the 
end  of  forty-eight  hours  is  usually  complete  in  plates  where  they  are 
numerous.  In  gelatin-stick  cultures  liquefaction  progresses  much  more 
rapidly  than  in  similar  cultures  of  the  cholera  spirillum,  and  a  stocking- 
shaped  pouch  of  liquefied  gelatin,  already  seen  after  forty-eight  hours, 
is  filled  with  a  cloudy  liquid.  The  liquefaction  increases,  and  in  twenty- 
four  hours  more  reaches  the  sides  of  the  tube  in  the  upper  part  of  the 
medium;  by  the  end  of  the  week  the  gelatin  is  usually  completely  lique- 
fied. Upon  the  surface  of  the  liquefied  medium  a  whitish  film  is  seen. 


406  BACTERIA  PATHOGENIC  TO  MAN 

Upon  agar  there  is  a  somewhat  more  luxuriant  growth  than  is  seen  with 
the  cholera  vibrio.  Upon  potato  this  spirillum  grows  at  room  tempera- 
ture and  produces  a  slimy,  grayish-yellow,  glistening  layer  which  soon 
extends  over  the  entire  surface.  The  cholera  spirillum  does  not  grow 
at  room  temperature,  and  in  the  incubator  produces  a  thin,  brownish 
layer.  The  absence  of  agglutination  with  a  suitable  dilution  of  the 
serum  of  an  animal  immunized  to  the  cholera  spirillum  is  a  valuable 
differential  sign. 

In  1884  Miller  observed  a  curved  bacillus  in  a  hollow  tooth,  which 
from  its  behavior  in  microscopic  preparations,  in  cultures,  and  animal 
experiments,  is  probably  identical  with  the  Finkler  and  Prior  spirillum. 
Very  similar  spirilla  have  been  found  by  others. 

Spirillum  of  Metchnikoff. 

Discovered  in  1888,  in  Odessa,  by  Gamaleia  in  the  intestinal  con- 
tents of  fowls  dying  of  an  infectious  disease,  which  prevails  in  certain 
parts  of  Russia  during  the  summer  months,  and  which  presents  symp- 
toms resembling  fowl  cholera.  Gamaleia's  experiments  show  that 
this  organism  is  the  cause  of  the  disease  mentioned.  It  has  since  been 
found  by  Pfuhl  and  Pfeiffer  in  the  water  of  the  Spree  at  Berlin,  and 
in  the  Lahn  by  Kutchler. 

Morphology. — Morphologically  this  spirillum  is  almost  identical 
with  the  cholera  spirillum.  In  the  blood  of  inoculated  pigeons  the 
diameter  is  sometimes  twice  as  great  as  that  of  the  cholera  spirillum, 
and  almost  coccus-like  forms  are  often  found.  A  single,  long,  undulat- 
ing flagellum  is  attached  to  one  end  of  the  spiral  filaments  or  curved 
rods. 

Stains  with  the  usual  aniline  colors,  but  not  by  Gram's  method. 

Cultural  Characters. — Upon  gelatin  plates  the  vibrio  Metchnikoff 
grows  considerably  faster  than  the  cholera  vibrio;  small,  white,  puncti- 
form  colonies  are  developed  at  the  end  of  twelve  hours;  these  rapidly 
increase  in  size  and  cause  liquefaction  of  the  gelatin  within  twenty- 
four  to  thirty  hours.  At  the  end  of  three  days  large,  saucer-like  areas 
of  liquefaction  may  be  seen,  the  contents  of  which  are  turbid,  as  a 
rule.  In  gelatin-stick  cultures  the  growth  is  almost  twice  as  rapid  as 
the  cholera  bacillus.  In  bouillon  at  37°  C.  development  is  very  rapid, 
and  the  liquid  becomes  clouded  and  opaque,  and  a  thin,  wrinkled  film 
forms  upon  the  surface.  On  the  addition  of  pure  sulphuric  acid  to 
twenty-four-hour  peptone  cultures  a  distinct  nitrosoindol  reaction  is 
produced.  Milk  is  coagulated  and  acquires  a  strongly  acid  reaction. 
The  spirillum  is  not  agglutinated  by  the  specific  cholera  agglutinin. 

Pathogenesis.  —  The  vibrio  Metchnikoff  is  pathogenic  for  fowls, 
pigeons,  and  guinea-pigs.  A  small  quantity  of  a  virulent  culture  fed 
to  chickens  and  pigeons  causes  their  death  with  the  local  and  general 
symptoms  of  fowl  cholera.  At  the  autopsy  the  most  constant  appear- 
ance is  hypersemia  of  the  entire  alimentary  canal.  A  grayish-yellow 
liquid,  more  or  less  mixed  with  blood,  is  found  in  considerable  quantity 


THE  CHOLERA  SPIRILLUM  AND  ALLIED  VARIETIES          407 

in  the  small  intestine.  In  the  watery  fluid  large  numbers  of  spirilla  are 
found.  A  few  drops  of  a  pure  culture  inoculated  subcutaneously  in 
pigeons  produce  septicaemia  and  cause  their  death  in  twelve  to  twenty- 
four  hours. 

In  contradistinction  to  the  pathogenic  virulence  of  these  spirilla 
for  pigeons  and  guinea-pigs,  the  cholera  spirillum  is  much  less  patho- 
genic. Pigeons  are  not  killed  by  the  intramuscular  inoculation  of  pure 
fresh  cultures  of  the  vibrio  cholera.  The  pathogenic  action  of  the 
vibrio  Metchnikoff  upon  pigeons  and  guinea-pigs,  producing  in  these 
animals  general  septicaemia  and  death,  is,  therefore,  a  characteristic 
point  of  difference  between  this  and  the  spirillum  of  Asiatic  cholera. 

Within  recent  years  numerous  other  spirilla,  the  so-called  "water 
vibrios,"  have  been  found  while  looking  for  the  cholera  spirillum. 


CHAPTER   XXXI. 

GLANDERS  BACILLUS  (BACILLUS  MALLEI). 

THIS  bacillus  was  discovered  and  proved  to  be  the  cause  of  glanders, 
by  isolation  in  pure  culture  and  communication  to  animals  by  inocu- 
lation, by  several  bacteriologists  almost  at  the  same  time  (1882) .  The 
bacilli  were  first  obtained  in  impure  cultures  by  Bouchard,  Capitan, 
and  Charrin,  and  first  accurately  studied  in  pure  culture  by  Loeffler 
and  Schiitz.  They  are  present  in  the  recent  nodules  in  animals  affected 
with  glanders,  and  in  the  discharge  from  the  nostrils,  pus  from  the 
specific  ulcers,  etc.,  and  occasionally  in  the  blood. 

Morphology. — Small  bacilli  with  rounded  or  pointed  ends,  from 
nutrient  agar  cultures,  Q.25/J.  to  0.5/*  broad  and  from  1.5/*  to  5/*  long; 
usually  single,  but  sometimes  united  in  pairs,  or  growing  out  to  long 
filaments,  especially  in  potato  cultures.  The  bacilli  frequently  break 
up  into  short,  almost  coccus-like  elements  (Fig.  126). 

Staining. — The  bacillus  mallei  stains  with  difficulty  with  the  aniline 
colors,  best  when  the  aqueous  solutions  of  these  dyes  are  made  feebly 
alkaline;  it  is  decolorized  by  Gram's  method.  This  bacillus  presents 
the  peculiarity  of  losing  very  quickly  in  decolorizing  solutions  the  color 
imparted  to  it  by  the  aniline-staining  solutions.  For  this  reason  it  is 
difficult  to  stain  in  sections.  Loeffler  recommends  his  alkaline  methy- 
lene-blue  solution  for  staining  sections,  and  for  decolorizing  a  mixture 
containing  10  c.c.  of  distilled  water,  2  drops  of  strong  sulphuric  acid, 
and  1  drop  of  a  5  per  cent,  solution  of  oxalic  acid;  thin  sections  to  be 
left  in  this  acid  solution  for  five  seconds. 

Biology. — An  aerobic,  non-motile  bacillus,  whose  molecular  move- 
ments are  so  active  that  they  have  often  been  taken  for  motility.  It 
grows  on  various  culture  media  at  37°  C.  Development  takes  place 
slowly  at  22°  C.  and  ceases  at  43°  C.  The  bacillus  does  not  form 
spores.  Exposure  for  ten  minutes  to  a  temperature  of  55°  C.,  or  for 
five  minutes  to  a  3  to  5  per  cent,  solution  of  carbolic  acid,  or  for  two 
minutes  to  a  1 : 5000  solution  of  mercuric  chloride,  destroys  its  vitality. 
As  a  rule,  the  bacilli  do  not  grow  after  having  been  preserved  in  a 
desiccated  condition  for  a  week  or  two;  in  distilled  water  they  are  also 
quickly  destroyed.  It  is  doubtful  whether  the  glanders  bacillus  finds 
conditions  in  nature  favorable  to  a  saprophytic  existence. 

Cultivation.  (For  methods  of  separation  see  page  411.) — It  grows 
well  in  the  incubating  oven  on  glycerin  agar.  Upon  this  medium  at 
the  end  of  twenty-four  to  forty-eight  hours,  whitish,  transparent  colonies 
are  developed,  which  in  six  or  seven  days  may  attain  a  diameter  of 
7  or  8  mm.  On  blood  serum  a  moist,  opaque,  slimy  layer  develops, 


GLANDERS  BACILLUS  409 

* 

which  is  of  a  yellowish-brown  tinge.  The  growth  on  cooked  potato 
is  especially  characteristic.  At  the  end  of  twenty-four  to  thirty-six 
hours  at  37°  C.  a  moist,  yellow,  transparent  layer  develops;  this  later 
becomes  deeper  in  color,  and  finally  takes  on  a  reddish-brown  color, 
while  the  potato  about  it  acquires  a  greenish-yellow  tint.  In  bouillon 
the  bacillus  causes  diffuse  clouding,  ultimately  with  the  formation  of  a 
more  or  less  ropy,  tenacious  sediment.  It  grows  on  media  possessing  a 
slightly  acid  reaction,  and  both  with  and  without  oxygen.  Milk  is 
coagulated  with  the  production  of  acid. 

Pathogenesis. — The  bacillus  of  glanders  is  pathogenic  for  a  number 
of  animals.  Among  those  which  are  most  susceptible  are  horses,  asses, 
guinea-pigs,  cats,  dogs,  ferrets,  moles,  and  field  mice;  sheep,  goats, 
swine,  rabbits,  white  mice,  and  house  mice  are  much  less  susceptible; 
cattle  are  immune.  Man  is  susceptible,  and  infection  not  infrequently 
terminates  fatallv. 


FIG.  126 


|  Glanders  bacilli.    Agar  culture.    X  1100  diameters. 

When  pure  cultures  of  the  bacillus  mallei  are  injected  into  horses 
or  other  susceptible  animals  true  glanders  is  produced.  The  disease 
is  characterized  in  the  horse  by  the  formation  of  ulcers  upon  the  nasal 
mucous  membrane,  which  have  irregular,  thickened  margins,  and 
secrete  a  thin,  virulent  mucus;  the  submaxillary  lymphatic  glands 
become  enlarged  and  form  a  tumor  which  is  often  lobulated;  other 
lymphatic  glands  become  inflamed,  and  some  of  them  suppurate  and 
open  externally,  leaving  deep,  open  ulcers;  the  lungs  are  also  involved, 
and  the  breathing  becomes  rapid  and  irregular.  In  farcy,  which  is  a 
more  chronic  form  of  the  disease,  circumscribed  swellings,  varying  in 
size  from  a  pea  to  a  hazel-nut,  appear  on  different  parts  of  the  body, 
especially  where  the  skin  is  thinnest;  these  suppurate  and  leave  angry- 
looking  ulcers  with  ragged  edges,  from  which  there  is  an  abundant 
purulent  discharge.  The  bacillus  of  glanders  can  easily  be  obtained 
in  pure  cultures  from  the  interior  of  suppurating  nodules  and  glands 


410  BACTERIA  PATHOGENIC  TO  MAN 

which  have  not  yet  opened  to  the  surface,  and  the  same  material  will 
give  successful  results  when  inoculated  into  susceptible  animals.  The 
discharge  from  the  nostrils  or  from  an  open  ulcer  may  contain  com- 
paratively few  bacilli,  and  these  being  associated  with  other  bacteria 
which  grow  more  readily  on  the  culture  media  than  the  bacillus  mallei, 
make  it  difficult  to  obtain  pure  cultures  from  such  material  by  the 
plate  method.  In  that  case,  however,  guinea-pig  inoculations  are 
useful. 

Of  test  animals  guinea-pigs  and  field  mice  are  the  most  susceptible. 
In  guinea-pigs  subcutaneous  injections  are  followed  in  four  or  five  days 
by  swelling  at  the  point  of  inoculation,  and  a  tumor  with  caseous  con- 
tents soon  develops;  then  ulceration  of  the  skin  takes  place,  and  a 
chronic  purulent  ulcer  is  formed.  The  essential  lesion  is  the  granulo- 
matous  tumor,  characterized  by  the  presence  of  numerous  lymphoid 
and  epithelioid  cells,  among  and  in  which  are  seen  the  glanders  bacilli. 
The  lymphatic  glands  become  inflamed  and  general  symptoms  of  infec- 
tion are  developed  in  from  two  to  four  weeks;  the  glands  suppurate 
and  in  males  the  testicles  are  involved;  finally  purulent  inflammation 
of  the  joints  occur,  and  death  ensues  from  exhaustion.  The  formation 
of  the  specific  ulcers  upon  the  nasal  mucous  membrane,  which  char- 
acterizes the  disease  in  the  horse,  is  rarely  seen  when  guinea-pigs  are 
inoculated.  In  these  the  process  is  often  prolonged,  or  remains  local- 
ized on  the  skin.  They  succumb  more  rapidly  to  intraperitoneal  injec- 
tion, usually  in  from  eight  to  ten  days,  and  in  males  the  testicles  are 
invariably  affected. 

MODE  OF  SPREAD. — Glanders  occur  as  a  natural  infection  only  in 
horses  and  asses;  the  disease  is  occasionally  communicated  to  man  by 
contact  with  affected  animals,  usually  by  inoculation  on  an  abraded 
surface  of  the  skin.  The  contagion  may  also  be  received  on  the  mucous 
membrane.  Infection  has  sometimes  been  produced  in  bacteriological 
laboratories.  In  man,  an  acute  and  chronic  form  of  glanders  may  be 
recognized,  and  an  acute  and  a  chronic  form  of  farcy.  The  disease  is 
fatal  in  about  60  per  cent,  of  the  cases.  It  is  transmissible  also  from 
man  to  man.  Washerwomen  have  been  infected  from  the  clothes  of 
a  patient.  The  infective  material  exists  in  the  secretions  of  the  nose, 
in  the  pus  of  glanders  nodules,  and  frequently  in  the  blood;  it  may 
occasionally  be  found  in  the  secretions  of  glands  not  yet  affected,  as  in 
the  urine,  milk,  and  saliva,  and  also  in  the  foetus  of  diseased  animals 
(Bonome).  From  recent  observations  it  appears  that  glanders  is  by  no 
means  an  uncommon  disease  among  horses,  particularly  in  southern 
countries,  sometimes  taking  a  mild  course  and  remaining  latent  for  a 
considerable  time.  Horses  apparently  healthy,  therefore,  may  possibly 
spread  the  disease. 

Attenuation  of  virulence  occurs  in  cultures  which  have  been  kept 
for  some  time,  and  inoculations  with  such  cultures  may  give  a  negative 
result,  or,  when  considerable  quantities  are  injected,  may  produce  a 
fatal  result  at  a  later  date  than  is  usual  when  small  amounts  of  a  recent 
culture  are  injected. 


GLANDERS  BACILLUS  411 

Immunity.— Attempts  have  been  made  to  produce  artificial  immu- 
nity against  glanders,  but  so  far  with  unsatisfactory  results.  According 
to  Strauss,  by  intravenous  inoculations  of  small  quantities  of  living 
bacilli,  dogs  may  be  protected  against  an  injection  of  quantities  which 
usually  kill  them.  Fenger  has  found  that  animals  inoculated  with 
glanders  bacilli  react  less  powerfully  to  fresh  injections;  and  that 
rabbits  which  have  recovered  from  an  injection  of  glanders  are  subse- 
quently immune,  the  immunity  lasting  for  from  three  to  six  weeks. 
Ladowski  has  obtained  positive  results  also  in  rabbits  and  cats  by 
intravenous  injections  of  sterilized  cultures.  Other  observers  have 
reported  not  only  the  production  of  immunity,  but  also  cures,  by  the 
use  of  mallein.  This  is  prepared  in  the  same  way  as  tuberculin.  It 
consists  of  the  glycerinated  bouillon  in  which  the  glanders  bacilli  have 
grown  and  which  contains  the  products  of  their  growth  and  activity. 
Concentrated  mallein  is  produced  by  evaporating  a  six-weeks-old 
culture  of  the  glanders  bacillus  in  5  per  cent,  glycerin  nutrient  veal 
bouillon  to  10  per  cent,  of  its  original  bulk.  Some  evaporate  the 
culture  fluid  only  to  20  per  cent.  The  dose  is  about  0.5  c.c.  of  the 
former,  or  2  c.c.  of  the  second  preparation. 

USE  OF  GUINEA-PIGS  AND  CULTURES  IN  DIAGNOSIS. — It  is  often 
difficult  to  demonstrate  microscopically  the  presence  of  the  bacillus 
of  glanders  in  the  nodules  which  have  undergone  purulent  degen- 
eration, in  the  secretions  from  the  nostrils,  or  in  the  pus  from  the 
specific  ulcers  and  suppurating  glands.  It  is  then  necessary  to  make 
immediate  cultures  and  also  animal  tests  of  these  discharges  by 
inoculating  susceptible  animals,  as  guinea-pigs  and  mice,  and  then 
from  these  to  obtain  a  pure  culture;  but  this  requires  time,  and  in 
clinical  work  it  is  of  great  importance  for  the  diagnosis  to  be  estab- 
lished as  quickly  as  possible.  With  this  view  Strauss  has  prepared  a 
method  which  is  prompt  and  which  has  given  very  satisfactory  results. 
This  consists  in  introducing  into  the  peritoneal  cavity  of  a  male  guinea- 
pig  some  material  or  a  culture  from  the  suspected  products.  If  it  be 
a  case  of  glanders,  the  diagnosis  may  be  made  within  two  or  three  days 
from  the  tumefaction  of  the  testicles,  which  become  red  and  swollen, 
and  show  evidences  of  pus  formation.  One  objection  to  this  method, 
however,  is  that  occasionally  from  the  injection  of  impure  material, 
as  in  the  nasal  secretion,  the  animal  may  die  of  septicaemia.  This  is 
particularly  frequent  when  field  mice  are  used  for  the  tests;  but  if  pure 
matter  can  be  obtained,  as  from  the  lymphatic  glands  of  the  horse, 
this  method  is  entirely  satisfactory. 

DIAGNOSTIC  USE  OF  MALLEIN. — The  diagnosis  of  glanders  in  horses, 
in  which  the  usual  symptoms  of  the  disease  have  not  yet  manifested 
themselves,  or  in  which  it  is  suspected,  may  often  be  made  by 
the  use  of  mallein.  Following  an  injection  of  mallein  in  a  glander- 
ous horse  (best  made  about  midnight)  there  will  be  a  local  reac- 
tion, and  a  general  reaction  with  a  rise  of  temperature.  The 
temperature  usually  begins  to  rise  three  or  four  hours  after  the 
injection,  and  reaches  its  maximum  between  the  tenth  and  twelfth 


412  BACTERIA  PATHOGENIC  TO  MAN 

hour.  Sometimes,  however,  the  highest  point  is  not  reached  until 
fifteen  or  eighteen  hours  after  the  injection.  This  elevation  of 
temperature  is  from  1.5°  to  2°  C.  (2°  to  3.5°  F.),  above  the  normal 
mean  temperature.  In  a  healthy  animal  the  rise  of  temperature,  as  a 
rule,  amounts  to  only  a  few  tenths  of  a  degree,  but  it  may  reach  1°  C. 
The  rise  of  temperature,  however,  should  be  considered  always  in 
connection  with  the  general  and  local  reactions.  In  a  glanderous 
animal,  after  an  injection  of  mallein,  the  general  condition  is  more  or 
less  profoundly  modified.  The  animal  has  a  dejected  appearance; 
the  countenance  is  pinched  and  anxious,  the  hair  is  rough,  the  flank  is 
retracted,  the  respirations  are  rapid,  there  are  often  rigors,  and  the 
appetite  is  gone.  In  healthy  animals  the  general  symptoms  do  not 
occur.  The  local  reaction  around  the  point  of  injection  in  a  glanderous 
animal  is  usually  very  marked.  A  few  hours  after  the  injection  there 
appears  a  large,  warm,  tense,  and  very  painful  swelling,  and  running 
from  this  will  be  seen  hot,  sensitive  lines  of  sinuous  lymphatics,  directed 
toward  the  neighboring  lymphatic  nodes.  This  oedema  increases  for 
twenty-four  to  thirty-six  hours  and  persists  for  several  days,  not  dis- 
appearing entirely  for  eight  or  ten  days.  In  healthy  animals,  at  the 
point  of  injection,  mallein  produces  only  a  small  oedematous  tumor,  and 
the  oedema,  instead  of  increasing,  diminishes  rapidly  and  disappears 
within  twenty-four  hours.  The  value  of  this  test  has  been  demon- 
strated by  numerous  experiments.  There  are  some  exceptions  to  the 
rule  as  described  above,  but  they  are  infrequent,  and  mallein  has  been 
used  with  considerable  success  as  a  diagnostic  aid  in  detecting  the 
existence  or  absence  of  glanders  in  doubtful  or  obscure  cases. 


CHAPTER  XXXII. 

THE  BACILLUS  OF  BUBONIC  PLAGUE— THE  BACILLUS  ICTEROIDES 
—THE  MICROCOCCUS  MELITENSIS. 

Bacillus  of  Bubonic  Plague  (Bacillus  Bacterium  Pestis  Bubonicse). 

HISTORICALLY  we  can  trace  the  bubonic  plague  back  to  the  third 
century.  In  Justinian's  reign  a  great  epidemic  spread  over  the  Roman 
empire  and  before  it  terminated  destroyed  in  many  portions  of  the 
country  nearly  50  per  cent,  of  the  people.  Among  the  most  fatal  forms 
of  infection  is  that  of  the  lungs.  Pneumonic  cases  are  not  alone  very 
serious,  but  they  readily  spread  infection.  The  bacillus  exciting  the 
disease  was  discovered  simultaneously  by  Kitasato  and  Yersin  (1894) 
during  an  epidemic  of  the  bubonic  plague  in  China.  It  is  found  in 
large  numbers  in  the  seropurulent  fluid  from  the  recent  buboes  char- 


FIG.  127  Fio.  128 


| 


• 

w 


Bacilli  froto  agar  culture.    X  1100  diam.  Bacilli  from  bouillon  culture.    X  1100  diam. 

icteristic  of  this  disease  and  in  the  lymphatic  glands  ;  more  rarely  in 
ihe  internal  organs  and  in  the  blood,  in  which  it  occurs  in  acute  hemor- 
rhagic  cases  and  shortly  before  death.  It  also  occurs  in  malignant 
cases  in  the  feces  of  men  and  animals.  The  bacillus  is  closely  allied 
to  the  hemorrhagic  septicaemia  group. 

Morphology.  —  The  bacilli  in  smears  from  acute  abscesses  or  infected 
tissues  are,  as  a  rule,  short,  thick  rods  with  rounded  ends.  The  central 
portion  of  the  bacillus  is  slightly  convex.  When  lightly  stained  the 
two  ends  are  more  colored  than  the  middle  portion.  The  bacilli  are 
mostly  single  or  in  pairs.  Bacilli  in  short  chains  occur  at  times.  The 


414 


BACTERIA  PATHOGENIC  TO  MAN 


length  of  the  bacilli  varies, but  on  the  average  is  about  1.6//  (1.5//  to  1.7/*), 
breadth  0.5/*  to  0.7/*.  Besides  the  usual  oval  form  the  plague  bacillus 
has  many  exceptional  variations  which  are  characteristic  of  it.  In 
smears  especially  from  old  buboes  one  looks  for  long  bacilli  with  clubbed 


FIG. 129 


Involution  forms  on  salt  agar.    (Kolle  and  Wassermann.) 


ends  (similar  to  involution  forms,  Fig.  129),  yeast-like  forms,  and 
bladder  shapes.  Some  of  these  stain  with  difficulty.  When  obtained 
from  cultures  the  bacilli  present  not  only  the  forms  already  mentioned, 
but  also  long  chains. 


FIG.  130 


Bacilli  in  acutely  inflamed  gland. 


Staining. — They  stain  readily  with  the  ordinary  aniline  dyes,  and 
especially  well  with  methylene  blue,  the  ends  being  usually  more  deeply 
colored  than  the  central  portion;  does  not  stain  by  Gram's  method. 


THE  BACILLUS  OF  BUBONIC  PLAQUE  415 

Biology. — An  aerobic,  non-motile  bacillus.  Grows  best  at  30°  to 
35°  C.  Does  not  form  spores.  Grows  on  the  usual  culture  media,  which 
should  have  a  slightly  alkaline  reaction.  Does  not  liquefy  gelatin. 
Grows  well  on  blood-serum  media.  It  grows  rapidly  on  glycerin  agar, 
forming  a  grayish-white  surface  growth.  The  bacilli  appear,  as  a  rule,  as 
short,  plump,  oval  bacilli,  but  a  few  present  elongated  thread  forms  which 
are  very  characteristic.  In  bouillon  a  very  characteristic  appearance 
is  produced,  the  culture  medium  remaining  clear  while  a  granular  or 
grumous  deposit  forms  on  the  walls  and  on  the  bottom  of  the  tube.  In 
bouillon  and  most  fluid  media  the  growth  is  in  the  form  of  short  or 
medium  chains  of  very  short,  oval  bacilli,  which  look  almost  like  strepto- 
cocci. 

Pathogenesis. — This  bacillus  is  pathogenic  for  rats,  mice,  guinea- 
pigs,  monkeys,  rabbits,  flies,  and  other  insects,  which  usually  die  within 
two  or  three  days  after  inoculation.  Then  at  the  point  of  inoculation 
is  found  a  somewhat  hemorrhagic  infiltration  and  oedema,  with  enlarge- 
ments of  the  neighboring  lymph  glands,  hemorrhages  into  the  peri- 
toneal cavity,  and  parenchymatous  congestion  of  the  organs.  The 
spleen  sometimes  shows  minute  nodules  resembling  miliary  tubercles. 
Microscopically  the  bacilli  are  found  in  all  the  organs  and  in  the  blood. 
The  disease  is  rapidly  communicated  from  one  animal  to  another,  and 
thus  its  extension  is  facilitated.  During  e'pidemics,  rats,  mice,  and 
flies,  in  large  numbers,  become  infected  and  die,  and  the  disease  is 
frequently  transmitted  through  them  to  man.  The  organism  is  found 
at  times  in  the  feces  of  sick  animals,  in  the  dust  of  infected  houses, 
and  in  the  soil. 

The  virulence  of  the  bacilli  in  cultures  and  in  nature  seems  to  vary 
considerably,  and  rapidly  diminishes  when  grown  on  artificial  media. 
The  growth  in  cultures  becomes  more  abundant  after  frequent  trans- 
plantation. The  virulence  of  the  organism  is  increased  by  successive 
inoculation  in  certain  animal  species,  and  then  its  pathogenic  properties 
for  other  species  are  less  marked. 

Yersin,  Calmette,  and  Borrel  have  succeeded  in  immunizing  animals 
against  the  bacillus  of  bubonic  plague  by  inoculation,  by  the  intravenous 
or  intraperitoneal  injection  of  dead  cultures,  or  by  repeated  subcu- 
taneous inoculation.  They  also  succeeded  in  immunizing  rabbits  and 
horses,  so  that  the  serum  afforded  protection  to  small  animals,  after  sub- 
cutaneous injection  of  virulent  cultures,  and  even  cured  those  which  had 
been  inoculated,  if  administered  within  twelve  hours  after  injection. 
The  serum  has  considerable  antitoxic  as  well  as  bactericidal  proper- 
ties. More  recently  this  serum  has  been  applied  to  the  treatment  of 
bubonic  plague  in  man,  with  promising  results.  Experience  has  shown 
that  the  treatment  is  more  efficacious  the  earlier  the  stage  of  the  dis- 
ease. When  treatment  is  begun  in  the  first  day  of  the  attack,  fever 
and  all  alarming  symptoms  frequently  disappear  with  astonishing 
rapidity.  In  cases  treated  at  a  later  stage  larger  doses  of  the  serum 
are  required,  and  even  in  the  favorable  cases  suppuration  of  the  buboes 
is  not  always  prevented.  In  some  of  the  early  cases  and  in  many  of 

or  rnr 


416  BACTERIA  PATHOGENIC  TO  MAN 

the  rather  late  ones  the  serum  fails.  When  the  disease  is  far  advanced 
the  serum  is  powerless.  For  immunizing  purposes  the  serum  should 
be  valuable,  and  a  single  injection  would  probably  give  protection  for 
several  weeks. 

Haffkine,  in  India,  has  recently  applied  his  method  of  preventive 
inoculation  to  the  bubonic  plague,  as  he  previously  did  with  cholera, 
and  apparently  with  equally  good  results.  This  method  consists  in  an 
inoculation  of  dead  cultures,  and  is  essentially  a  protective  rather  than 
a  curative  treatment.  It  gives  after  six  to  ten  days  a  considerable 
immunity,  lasting  a  month  or  more.  By  means  of  these  two  methods 
of  inoculation,  along  with  strict  quarantine  regulations,  it  is  to  be  hoped 
that  this  disease  which  under  the  name  of  Black  Death  once  decimated 
the  populations  of  the  earth,  and  which  in  the  East  still  causes  great 
mortality  at  times,  may  finally  be  greatly  restricted. 

Duration  of  Life  Outside  of  the  Body. — In  cultures  protected  from  the 
air  and  light  the  plague  bacilli  may  live  one  year  or  more.  In  the  bod  es 
of  dead  rats  they  may  live  for  two  months.  In  sputum  from  pneu- 
monic cases  the  bacilli  lived  ten  days.  Upon  sugar  sacks,  food,  etc., 
they  may  live  six  to  fifteen  days. 

Resistance  to  Deleterious  Influences. — The  bacilli  resemble  the  colon 
bacilli  in  their  reaction  to  heat  and  disinfectants.  Boiling  for  one  to 
two  minutes  kills  them.  Carbolic  acid,  5  per  cent,  solution,  kills  cul- 
ture in  one  minute,  in  2^  per  cent,  in  two  minutes,  etc. 

Bacteriological  Diagnosis. — When  the  lymph  glands  are  acutely  in- 
flamed but  not  yet  suppurated  cut  down  on  one  and  make  cultures  on 
nutrient  agar  slanted  in  tubes.  If  pus  has  formed  withdraw  a  little  by 
means  of  the  hypodermic  needle.  There  should  also  be  made  smears 
from  the  suspected  bubo,  or  in  case  of  pneumonia  from  the  sputum.  If 
the  patient  is  dead,  cultures  from  the  spleen  and  heart's  blood  are  also 
taken  when  possible.  Suspected  animals,  such  as  rats  and  mice,  when 
freshly  killed,  are  examined  as  in  man;  when  decomposed,  rats  and 
guinea-pigs  should  be  inoculated. 

Bacillus   Icteroides. 

In  1897  Sanarelli  announced  the  discovery  of  a  micro-organism 
which  he  claimed  to  be  the  specific  cause  of  yellow  fever.  This  he 
called  the  "bacillus  icteroides."  It  is  found  in  the  circulating  blood 
and  in  the  tissues  of  most  yellow  fever  patients. 

Morphology. — It  resembles  the  colon  bacilli  in  many  characteristics. 
The  work  of  Reed  and  his  associates  having  thoroughly  overthrown  the 
claims  of  Sanarelli,  its  description  is  omitted  with  the  exception  of  a  few 
notes. 

It  stains  readily  with  the  ordinary  aniline  dyes,  but  not  by  Gram's 
method. 

Biology. — A  motile,  facultative,  anaerobic,  non-liquefying  bacillus. 
Does  not  form  spores  as  far  as  known.  Grows  readily  in  all  the  ordinary 
culture  media  at  the  room  temperature,  but  best  at  37°  C.  in  the  incu- 


BA  GILL  US  ICTEROIDES  4 1 7 

bator.  Cultures  on  agar  at  20^  C.  are  characteristic,  according  to 
Sanarelli.  Grown  at  room  temperature  they  appear  like  drops  of  milk, 
opaque,  projecting,  and  with  pearly  reflections. 

The  bacillus  icteroides  ferments  glucose  and  saccharose,  but  does 
not  coagulate  milk. 

Pathogenesis. — It  is  pathogenic  for  the  greater  number  of  the  domes- 
tic animals;  but  birds  are  completely  refractory. 


CHAPTEE   XXXIII. 

REPRESENTATIVE  PATHOGENIC  MICRO-ORGANISMS  BELONGING 
TO  THE  HIGHER  BACTERIA. 

THE  members  of  the  higher  bacteria  which  are  pathogenic  to  man 
have  as  yet  been  incompletely  studied  and  classified.  The  following 
divisions  serve  as  an  attempt  at  differentiation: 

1.  Actinomyces  is  characterized  by  the  radiating  wreath-like  forms 
which  it  alone  produces  in  the  living  body. 

2.  Streptothrix,  by  its  abundant  true  branching,  wavy  growth,  later 
fragmentation,  and  formation  of  conidiae,  which   serve   as  organs  of 
propagation,  and  in  this  sense  may  be  considered  as  spores. 

3.  Cladothrix,  by  its  false  branching,  rapid  fragmentation,  and  then 
bacillary  characteristics  in  old  cultures. 

4.  Leptotkrix,  by  its  lack  of  observed  branching,  non-wavy  growth, 
but,  on  the  contrary,  stiff,  almost  straight  threads,  in  which  division 
processes  are  seldom  or  never  observed. 

These  higher  bacteria  may  rightly  be  considered,  according  to  their 
development,  as  a  transition  group  between  the  simple  bacteria  and 
the  more  highly  developed  fungi. 

The  streptothrix  group  of  micro-organisms  while  having  many 
affinities  with  the  bacteria,  yet  differs  from  them  in  many  important 
respects  which  link  them  with  the  fungi.  They  develop  from  spore- 
like  bodies  into  cylindrical  dichotomously  branching  threads,  which 
grow  into  colonies,  the  appearance  of  which  suggests  a  mass  of  radi- 
ating filaments.  Under  favorable  conditions  certain  of  the  threads 
become  fruit  hyphse,  and  these  break  up  into  chains  of  round,  spore- 
like  bodies,  which  do  not,  however,  have  the  same  staining  reac- 
tions nor  resisting  powers  as  true  spores.  The  tubercle  grass  and 
diphtheria  bacilli  are  by  some  believed  to  properly  belong  in  the  strep- 
tothrix group,  on  account  of  the  true  branching  forms  developed  by 
them  under  certain  conditions.  The  actinomyces  fungus  is  by  some 
classed  in  the  streptothrix  group. 

The  Micro-organism  of  Actinomycosis. 

This  parasite  was  first  discovered  by  Bollinger  in  the  ox  and  given 
the  name  of  actinomyces,  or  ray  fungus,  by  the  botanist  Harz.  The 
two  most  important  publications  on  the  subject  of  the  biology  of  this 
micro-organism  are  those  of  Bostroem  and  of  Wolf  and  Israel,  published 
in  1890  and  1891,  respectively. 


MICRO-ORGANISMS  BELONGING  TO  THE  HIGHER  BACTERIA     419 

The  characteristics  of  the  micro-organism  described  by  these  workers 
differed  greatly  and  have  led  to  confusion.  Bostroem's  organism  grew 
best  aerobically  and  developed  well  at  room  temperature.  He  noted 
the  intimate  relation  of  the  organism  with  fragments  of  grain,  and  this 
led  to  the  finding  of  similar  micro-organisms  in  the  outer  world  on  grains, 
grasses,  etc. 

There  is  no  doubt  that  some  suppurative  processes  have  been  due 
to  organisms  of  these  characteristics,  but  they  do  not  seem  to  excite 
true  actinomycosis. 

Wolf  and  Israel  described  a  micro-organism  from  two  human  cases, 
which  differ  from  that  described  by  Bostroem,  but  agrees  with  the  micro- 
organisms obtained  by  most  of  the  more  recent  investigators.  It  grew 
best  under  anaerobic  conditions  and  did  not  grow  at  room  temperature. 
Its  growth  was  much  less  luxuriant  than  Bostroem's  micro-organism. 
On  the  surface  of  anaerobic  agar  slant  cultures  on  the  third,  fourth,  and 
fifth  day  numerous  minute  isolated  dew-drop-like  colonies  appeared,  the 
largest  pinhead  in  size.  These  gradually  became  larger  and  formed 
ball-like,  irregularly  rounded  elevated  nodules  varying  in  size  up  to 
that  of  a  millet-seed,  exceptionally  attaining  the  size  of  a  lentil  or  larger. 
As  a  rule  the  colonies  did  not  become  confluent,  and  an  apparently 
homogeneous  layer  of  growth  was  seen  to  be  made  up  of  separate 
nodules  if  examined  with  a  lens.  In  some  instances  the  colonies  pre- 
sented a  prominent  centre  with  a  tabulated  margin  and  appeared  as 
rosettes.  A  characteristic  of  the  colonies  was  that  they  sent  into  the  agar 
root-like  projections.  In  aerobic  agar  slant  cultures  no  growth  or  a  slow 
and  very  feeble  growth  was  obtained.  In  stab  cultures  the  growth  was 
sometimes  limited  to  the  lower  portion  of  the  line  of  inoculation  or  was 
more  vigorous  there.  In  bouillon,  after  three  to  five  days,  growth 
appeared  as  small  white  flakes,  partly  floating  and  partly  collected  at 
the  bottom  of  the  tube.  Growth  occurred  in  bouillon  under  aerobic 
conditions,  but  was  better  under  anaerobic  conditions.  The  micro- 
organism in  smear  preparations  from  agar  cultures  appeared  chiefly  as 
short  homogeneous,  usually  straight,  but  also  comma-like  or  bowed 
rods,  whose  length  and  breadth  varied.  In  many  cultures  short  clump 
rods  predominated,  and  in  others  longer,  thicker,  or  thinner  individuals 
were  more  numerous.  The  ends  of  the  rods  often  showed  olive  or  ball- 
like  swellings.  Some  twenty  guinea-pigs  and  rabbits  were  inoculated, 
most  of  them  in  the  peritoneal  cavity,  with  pieces  of  agar  culture. 
Eighteen  animals  were  killed  after  four  to  seventeen  weeks,  and  four 
were  still  alive  seven  to  nine  months  after  the  inoculation.  Seventeen 
rabbits  and  one  guinea-pig  showed  at  the  autopsy  tumor  growths  mostly 
in  the  peritoneal  cavity  and  in  one  instance  in  the  spleen.  In  the  four 
animals  still  living  tumors  were  to  be  felt  in  the  abdominal  wall.  The 
tumors  in  the  peritoneal  cavity  were  millet-seed  to  plum  size,  and  were 
situated  partly  on  the  abdominal  wall  and  partly  on  the  intestines,  the 
omentum,  the  mesentery,  and  in  the  liver  or  in  adhesions.  While  the 
surface  of  the  smaller  tumors  was  always  smooth,  the  surface  of  the 
larger  tumors  showed  small  hemispherical  prominences,  giving  them  the 


420 


BACTERIA  PATHOGENIC  TO  MAN 


appearance  of  conglomerates  of  smaller  tumors.  On  section  the  larger 
tumors  presented  a  tough  capsule  from  which  anastomosing  septa 
extended  inward  enclosing  cheesy  masses.  Microscopic  examination 
of  the  tumors  showed  in  all  cases  but  one  the  presence  of  typical  actino- 
myces  colonies,  in  most  cases  with  typical  "clubs."  The  general 
histological  appearance  of  the  tumors  was  like  that  of  actinomycotic 
tissue. 

Wolff  in  a  later  paper  reports  that  an  animal  inoculated  in  the  peri- 
toneal cavity  with  a  culture  of  the  same  organism  had  lived  a  year  and  a 
half.  At  the  autopsy  several  tumors  were  found  in  the  peritoneal  cavity, 
and  in  the  liver  a  large  typical  tumor  in  which  were  many  colonies  which 
by  microscopic  examination  were  shown  to  be  typical  club-bearing 
actinomyces  colonies. 

Naked-eye  Appearance  of  Colonies  of  Parasite  in  Tissues. — In  both  man 
and  animals  they  can  be  readily  seen  in  the  pus  from  the  affected 


FIG.  131 


A  typical  "  club  "-bearing  colony  of  actinomyces.    X  325  diameters.    (From  Wright.) 


regions  as  small,  white,  yellowish  or  greenish  granules  of  pinhead  size 
(from  0.5  to  2  mm.  in  diameter).  When  pus  has  not  formed  they  lie 
embedded  in  the  granulation  tissue. 

Microscopic  Appearance. — Microscopically  these  bodies  are  seen  to  be 
made  up  of  threads,  which  radiate  from  a  centre  and  present  bulbous, 
club-like  terminations  (Fig.  131).  These  club-like  terminations  are 
characteristic  of  the  actinomyces.  They  are  generally  arranged  in 
pairs,  closely  crowded  together,  and  are  very  glistening  in  appearance. 
The  threads  which  compose  the  central  mass  of  the  granules  are  from 
0.3/^  to  0.5,"  in  diameter;  the  clubs  are  from  6//  to  8ft  in  diameter. 

The  organism  is  stained  with  the  ordinary  aniline  colors,  also  by 
Gram's  solution;  when  stained  with  gentian  violet  and  by  Grain's 
method  the  threads  appear  more  distinct  than  when  stained  with 
methylene  blue.  The  clubs  lose  their  stain  by  Gram's  method  and 
take  the  contrast  stain. 


MICRO-ORGANISMS  BELO\<,I\<;  TO  THE  HIGHER  BACTERIA     421 

Isolation  of  Actinomyces.—  Wright1  recommends  that  granules, 
preferably  obtained  from  closed  lesions,  are  first  thoroughly  washed  in 
sterile  water  or  bouillon  and  then  crushed  between  two  sterile  glass 
slides.  In  bovine  cases  make  sure  the  granule  has  filamentous  masses, 
for  if  not  no  culture  will  grow.  The  crushed  granule  is  transferred 
to  a  tube  of  melted  1  per  cent,  glucose  agar  at  40°  C.  The  material 
is  thoroughly  distributed  by  shaking  and  the  tube  placed  in  the  incu- 
bator. A  number  of  granules  after  washing  should  be  placed  on  the 
inside  of  a  sterile  test-tube  and  allowed  to  dry.  In  this  way,  should  the 
material  be  contaminated,  the  drying  of  the  granules  for  several  weeks 
may  kill  off  the  other  bacteria.  The  tube  should  be  examined  daily. 
If  a  number  of  living  filaments  were  added  to  the  agar  a  large  number 
of  colonies  will  develop.  These  will  be  most  numerous  in  the  depth  in 
a  zone  five  to  twelve  millimetres  below  the  surface. 

From  this  primary  culture  a  colony  is  cut  out  and  the  bit  of  agar 
washed  in  bouillon  and  then  inserted  in  a  tube  of  melted  agar.  The 
growth  in  this  will  give  material  for  transplants. 

•Is  actinomycosis  due  to  a  single  micro-organism  or  to  a  group  of 
organisms  having  widely  different  characteristics? 

Wright2  has  recently  made  an  important  research  study  on  this 
question.  His  conclusions  were  as  follows :  From  thirteen  human  cases 
and  from  two  in  cattle  the  organisms  seem  to  be  all  of  one  species,  for 
the  differences  among  the  various  strains  are  no  greater  than  among 
various  strains  of  tubercle  or  diphtheria  bacilli. 

This  micro-organism  grows  well  only  in  agar  and  bouillon  cultures 
and  in  the  incubator;  in  the  other  usual  culture  media  and  at  room 
temperature  it  grows  only  very  little  or  not  at  all.  It  is  essentially  an 
anaerobe.  It  does  not  form  spore-like  reproductive  elements.  In 
cultures  its  colonies  are  similar  in  character  to  colonies  of  the  micro- 
organism in  the  lesions  of  actinomycosis.  If  colonies  of  the  micro- 
organism are  immersed  in  animal  fluids,  such  as  blood  serum  and  serous 
pleuritic  fluid,  the  filaments  of  the  colonies  in  immediate  contact  with 
the  fluid  may,  under  certain  unknown  conditions,  become  invested  with 
a  layer  of  hyaline  eosin-staining  material  of  varying  thickness,  and  the 
filament  may  then  disappear.  Thus  structures  are  produced  that  seem 
to  be  identical  with  the  characteristic  "clubs"  of  actinomyces  colonies 
in  the  lesions. 

Inoculation  experiments  on  animals  were  made  with  the  cultures  of 
the  micro-organism  from  thirteen  cases,  including  the  two  bovine  cases. 
All  of  these  strains  were  found  to  be  capable  of  forming  the  characteristic 
"club"-bearing  colonies  in  the  tissues  of  the  experimental  animals. 
These  colonies  were  either  enclosed  in  small  nodules  of  connective  tissue 
or  were  contained  in  suppurative  foci  within  nodular  tumors  made  up 
of  connective  tissue  in  varying  stages  of  development.  With  the  cultures 
from  most  of  the  cases  nodular  lesions  identical  in  histological  character 
with  those  of  actinomycosis  were  produced  in  inoculated  animals  and 

i  Journal  of  Medical  Research,  May,  1905.  2  Loc.  cit. 


422  BACTERIA  PATHOGENIC  TO  MAN 

with  some  of  the  cultures  relatively  extensive  lesions,  considering  the 
size  of  the  animal.  The  most  extensive  lesions  showed  little  progressive 
tendency,  and  in  only  a  very  few  instances  did  multiplication  of  the 
micro-organism  in  the  body  of  the  inoculated  animal  seem  probable. 
In  view  of  the  negative  or  ambiguous  results  of  those  who  have  inoculated 
healthy  animals  with  actinomyces  directly  from  the  lesions,  it  would 
seem  that  the  results  of  the  inoculation  of  animals  with  the  cultures 
described  in  this  paper  afford  as  much  proof  as  can  be  expected  from 
such  experiments  that  the  micro-organism  in  the  cultures  was  identical 
with  the  micro-organism  in  the  original  lesions. 

I  do  not  accept  the  prevalent  belief,  based  on  the  work  of  Bostroem, 
Gasperini,  and  others,  that  the  specific  infectious  agent  of  actinomycosis 
is  to  be  found  among  certain  branching  micro-organisms,  widely  dis- 
seminated in  the  outer  world,  which  differ  profoundly  from  actinomyces 
bo  vis  in  having  spore-like  reproductive  elements.  I  think  that  these  should 
be  grouped  together  as  a  separate  genus  with  the  name  of  nocardia,  and 
that  those  cases  of  undoubted  infection  by  them  should  be  called  nocard- 
iosis  and  not  actinomycosis.  The  term  actinomycosis  should  be  used 
only  for  those  inflammatory  processes  the  lesions  of  which  contain  the 
characteristic  granules  or  "drtisen."  That  a  nocardia  ever  forms  these 
characteristic  structures  in  lesions  produced  by  it  in  man  or  cattle  has 
not  been  convincingly  shown. 

Because  the  micro-organism  here  described  does  not  grow  well  on  all 
the  ordinary  culture  media  and  practically  not  at  all  at  room  temperature, 
I  do  not  believe  that  it  has  its  usual  habitat  outside  of  the  body.  It 
seems  to  me  very  probable  that  it  is  a  normal  inhabitant  of  the  buccal 
cavity  and  gastrointestinal  tract. 

The  cultures  are  quite  resistant  to  outside  influences;  dried,  they 
may  be  kept  for  a  year  or  more;  they  are  killed  by  an  exposure  of  five 
minutes  to  a  temperature  of  75°  C. 

Occurrence  in  Animals. — Actinomycosis  is  quite  prevalent  among 
cattle,  in  which  it  occurs  endemically;  it  is  more  rare  among  swine  and 
horses,  and  is  sometimes  found  in  man.  The  disease  is  rarely  com- 
municated from  one  animal  to  another  and  no  case  is  known  where  a 
direct  history  of  human  contagion  has  been  obtained.  The  cereal 
grains,  which  from  their  nature  are  capable  of  penetrating  the  tissues, 
have  been  repeatedly  found  in  centres  of  actindmycotic  infection.  This 
usually  occurs  in  the  vicinity  of  the  mouth,  where  injuries  have  been 
accidentally  caused.  The  micro-organism  may  also  be  introduced  by 
means  of  carious  teeth.  Cutaneous  infection  has  been  produced  by 
wood  splinters,  and  infection  of  the  lungs  by  aspiration  of  fragments 
of  teeth  containing  the  fungus.  The  presence  of  the  micro-organism  in 
cereal  grains,  which  was  formerly  accepted,  is  denied  by  Wright  and 
therefore  certainly  placed  in  doubt.  The  further  distribution  of  the 
fungus  after  it  is  introduced  into  the  tissues  is  effected  partly  by  its 
growth  and  partly  by  conveyance  by  means  of  the  lymphatics  and 
leukocytes.  Not  infrequently  a  mixed  infection  with  the  pyogenic 
cocci  occurs  in  actinomycosis. 


MICRO-ORGANISMS  BELONGING  TO  THE  HIGHER  BACTERIA     423 

In  the  earliest  stages  of  its  growth  the  parasite  gives  rise  to  a  small 
granulation  tumor,  not  unlike  that  produced  by  the  tubercle  bacillus, 
which  contains,  in  addition  to  small  round  cells,  epithelial  elements 
and  giant  cells.  After  it  reaches  a  certain  size  there  is  great  proliferation 
of  the  surrounding  connective  tissue,  and  the  growth  may,  particularly 
in  the  jaw,  look  like,  and  was  long  mistaken  for,  osteosarcoma.  Finally, 
suppuration  occurs,  which,  according  to  Israel,  may  be  produced 
directly  by  the  fungus  itself. 

The  experimental  production  of  actinomycosis  in  animals  has  been 
followed  by  negative  or  very  unsatisfactory  results.  When  artificially 
introduced  into  the  tissues  the  organism  is  either  absorbed  or  encap- 
sulated. If  introduced  in  large  quantities  multiple  nodules  are  appar- 
ently formed  in  some  cases,  which  may  suggest  the  production  of  a 
general  infective  process;  but  on  closer  inspection  of  these  nodules 
the  thread-like  portion  of  the  fungus  is  found  to  have  disappeared, 
leaving  only  the  remains  of  the  club-like  ends,  thus  showing  that  no 
growth  has  taken  place.  Ponfick,  Johne,  Rotter,  Liming,  and  Hanan 
claim  to  have  obtained  positive  results  in  animals,  but  according  to 
Bostrcem  these  results  are  not  conclusive.  The  animals  used  for  experi- 
mentation have  been  calves,  swine,  dogs,  rabbits,  and  guinea-pigs,  the 
places  of  inoculation  being  the  anterior  chamber  of  the  eye,  the  sub- 
cutaneous intercellular  tissue,  the  peritoneum,  and  the  blood,  and  the 
material  employed  for  inoculation  being  pus  from  the  infected  regions 
in  animals  and  man,  very  rarely  cultures. 

Streptothrix  Infections. — From  widely  scattered  localities  and  at  long 
intervals  of  time  reports  have  been  published  describing  unique  cases 
of  disease  produced  by  varieties  of  micro-organisms  belonging  to 
the  genus  Streptothrix.  In  some  of  these  cases  points  of  similarity 
can  be  recognized  in  the  clinical  symptoms  and  the  gross  patho- 
logical lessons,  while  others  differ  widely  in  both  respects.  They  have 
been  found  in  brain  abscess,  cerebrospinal  meningitis,  pneumonic  areas, 
and  in  other  pathological  conditions.  Eppinger  injected  cultures 
into  guinea-pigs  and  rabbits,  and  observed  that  it  caused  a  typical 
pseudotuberculosis.  Consolidation  of  portions  of  both  lungs,  thicken- 
ing of  the  peritoneum,  and  scattered  nodules  resembling  tubercles, 
were  noted  in  a  case  of  human  infection  as  due  to  a  Streptothrix  by 
Flexner,  in  which  the  pathological  picture  of  the  disease  resembled 
so  nearly  tuberculosis  in  human  beings  that  the  two  diseases  could  be 
separated  only  by  the  causative  micro-organism  in  each  case.  But  in 
no  two  cases  reported  up  to  the  present  time  have  the  descriptions 
of  the  micro-organisms  found  agreed  in  all  particulars.  In  some 
cases  no  attempt  at  cultivation  was  made.  In  other  cases  numerous 
and  careful  plants  on  various  culture-media  failed  to  develop  the 
specific  organism.  In  the  remaining  cases  in  which  the  Streptothrix 
was  obtained  in  pure  culture,  the  descriptions  of  the  growth  charac- 
teristics essentially  differ.  In  a  recent  review  of  the  literature  Tuttle 
was  able  to  find  the  reports  of  only  twelve  cases  in  which  a  Streptothrix 
was  found  in  sufficient  abundance  to  have  been  an  important,  if  not  the 


424  BACTERIA  PATHOGENIC  TO  MAN 

principal,  factor  in  producing  disease.  These  cases  were  all  fatal,  and 
only  once  was  the  character  of  the  disease  recognized  during  life.  As 
the  clinical  symptoms  and  the  lesions  in  the  human  subject  as  well  as  in 
the  animals  experimentally  inoculated  with  the  streptothrix  often  resem- 
ble those  of  miliary  tuberculosis,  so  that  a  number  of  these  cases  have 
been  reported  as  pseudotuberculosis,  the  question  is  naturally  suggested 
whether  such  cases  of  streptothrix  tuberculosis  are  not  more  numerous 
than  the  few  reported  cases  would  indicate.  The  almost  universal 
prevalence  of  genuine  tuberculosis  and  the  extreme  gravity  of  the  dis- 
ease have  so  long  occupied  the  attention  and  study  of  the  medical  pro- 
fession that  much  is  taken  for  granted,  and  cases  in  which  the  symp- 
toms and  lesions  resemble  with  some  closeness  those  characteristic  of 
the  well-known  disease  may  easily  be  set  down  without  question  to 
the  account  of  the  tubercle  bacillus.  The  cases  of  streptothrix  tuber- 
culosis so  far  reported  have  all  been  fatal,  and  the  lesions  for  the  most 
part  have  been  widely  distributed,  but  in  a  number  of  cases  old  lesions 
have  been  found  which  suggest  that  the  disease  may  have  been  localized 
for  a  longer  or  shorter  time,  and  then,  by  some  accident,  may  have 
become  rapidly  general.  In  this  respect,  also,  these  cases  may  resemble 
tuberculosis.  Whether  all  cases  of  streptothrix  disease  in  the  human 
subject  are  general  and  fatal,  or,  as  in  tuberculosis  and  actinomycosis, 
there  may  be  cases  of  localized  disease  which  recover,  are  questions 
which  have  not  been  decided  at  the  present  time.  The  methods  em- 
ployed to  demonstrate  the  presence  of  tubercle  bacilli  render  the  strepto- 
thrices  invisible.  Again,  unless  the  observer  keeps  in  mind  the  possi- 
bility of  streptothrix  infection,  he  may  not  appreciate  the  importance 
of  the  slender  threads  with  or  without  branches,  and  may  consider 
them  accidental  bacilli,  or  varieties  of  leptothrix  or  non-pathogenic 
fungi.  As  the  lungs  have  appeared  to  be  the  seat  of  the  primary 
infection  in  most  of  the  cases  of  human  streptothrix  disease,  it  is  very 
desirable  that  all  cases  presenting  the  physical  signs  of  tuberculosis, 
in  which  repeated  examinations  fail  to  discover  the  tubercle  bacillus, 
should  be  systematically  examined  for  streptothrix  threads.  In  this 
way  alone  can  the  frequency  of  the  disease  be  determined.  Gram's 
method  of  staining  or  the  Ziehl-Neelson  solution  decolorized  with 
aniline  oil  seem  to  be  the  most  reliable  agents  for  demonstrating  these 
organisms.  The  streptothrices  are  widely  distributed  and  are  not  very 
infrequently  met  with,  but  as  yet,  with  the  exceptions  mentioned  above, 
very  little  is  known  about  them.  Kruse  mentions  nineteen  varieties, 
including  the  actinomyces.  Some  of  them  are  non-pathogenic;  some 
are  pathogenic  for  certain  animals,  and  others  are  pathogenic  for  both 
man  and  animals. 

In  studying  the  descriptions  of  the  different  varieties  of  these  micro- 
organisms, it  seems  that,  as  in  the  case  of  certain  bacteria,  different 
observers  may  possibly  have  described  the  same  variety  under  different 
names. 

Ttittle's  report  of  the  case  of  general  streptothrix  infection  at  the 
Presbyterian  Hospital,  gives  such  a  good  clinical,  bacteriological,  and 


MICRO-ORGANISMS  BEL(>.\',I  \<;  TO  THE  HIGHER  BACTERIA     425 

pathological  picture  of  a  case  of   this   infection    that  a   considerable 
portion  of  it  is  repeated  here: 

Six  days  before  her  admission  to  the  hospital  her  illness  began  with 
a  severe  chill,  and  fever,  and  pain  in  her  left  side  and  back.  The  fol- 
lowing day  the  pain  in  the  side  was  worse  and  breathing  was  difficult. 
She  began  to  cough  and  had  some  expectoration,  but  no  blood 
noticed  in  the  sputa.  At  irregular  intervals  she  had  alternating  hot 
and  chilly  sensations. 

On  admission,  the  patient  complained  of  pain  in  the  left  side  of  the 
chest,  cough,  fever,  weakness,  and  prostration.  Her  temperature  was 
103°,  and  her  pulse  and  respirations  were  rapid. 

Physical  Examination. — The  patient  is  poorly  nourished  and  ana-mic. 
Lungs:  Anteriorly  the  right  lung  is  normal.  On  the  left  side,  over  an 
area  two  inches  by  three  inches  just  to  the  left  of  the  nipple,  there  is 
marked  dulness  to  percussion,  and  bronchial  voice  and  breathing.  No 
rales  are  heard.  Posteriorly,  the  left  lung  is  normal.  At  the  extreme 
base  of  the  right  lung  there  is  slight  dulness  and  diminished  breathing, 
with  crepitant  rales  after  coughing.  The  voice  is  normal. 

The  history  of  the  disease  and  the  physical  signs  indicated  an  attack 
of  acute  lobar  pneumonia,  the  area  of  consolidation  being  small  and 
situated  in  the  lower  part  of  the  left  upper  lobe  in  front.  Frequent 
and  violent  coughing,  with  almost  no  expectoration,  pain  in  the  affected 
side  and  in  the  lumbar  region,  restlessness  and  sleeplessness,  and 
involuntary  urination  were  the  symptoms  noted  during  the  first  four 
days  in  the  hospital.  The  pneumonic  area  increased  somewhat,  and 
extended  backward  to  the  posterior  axillary  line,  and  the  temperature 
was  continuous  at  103°  to  103.5°.  On  the  fifth  day  the  temperature 
fell  two  degrees,  and  signs  of  resolution  appeared  in  the  consolidated 
area.  The  apparent  improvement,  however,  was  of  short  duration. 
On  the  sixth  day  the  temperature  rose  to  104.5°,  and  continued  to 
rise  each  day,  reaching  107.5°  shortly  before  death,  which  occurred 
on  the  ninth  day  in  the  hospital  and  the  fifteenth  day  of  the  disease. 
During  the  last  four  days  the  patient  complained  bitterly  of  pain  in  the 
lumbar  region  and  in  the  thighs  and  legs,  and  of  intense  vesical  tenesmus. 
The  stools  and  urine  were  passed  involuntarily.  Signs  of  consolidation 
were  found  in  the  right  lower  lobe,  behind.  There  were  repeated  attacks 
of  profuse  sweating.  On  the  day  before  her  death  three  indurated 
swellings  beneath  the  skin  were  noticed.  One,  on  the  left  forearm, 
about  the  size  of  a  walnut,  apparently  contained  pus.  Two,  of  smaller 
size,  were  situated  in  the  right  groin. 

Blood  cultures  from  a  vein  in  the  arm,  taken  on  the  sixth  day,  re- 
mained sterile.  Subsequent  attempts  failed  on  account  of  the  feeble 
circulation.  The  leukocyte  count  on  the  seventh  day  was  36,000. 

Autopsy. — On  the  right  arm,  the  left  forearm,  the  abdominal  wall, 
and  on  both  thighs  there  are  eight  or  ten  slightly  projecting,  roundel, 
fluctuating,  subcutaneous  swellings  from  one-half  inch  to  one  inch  in 
diameter.  The  skin  over  most  of  these  nodules  is  unaltered,  but  over 
the  larger  ones  there  is  a  slight  bluish  discoloration.  The  nodules  were 


426  BACTERIA  PATHOGENIC  TO  MAN 

found  to  be  collections  of  bluish-gray,  thick,  mucilaginous  matter,  which 
is  very  tenacious  and  can  be  drawn  out  into  long  threads.  The  pericar- 
dium is  normal.  The  valves  are  normal.  In  the  wall  of  the  right  ventricle 
are  several  small  white  areas  which  look  like  septic  infarctions,  and 
one  of  larger  size  in  the  wall  of  the  left  ventricle  corresponds  with  the 
position  of  a  thrombus,  and  apparently  was  the  exciting  cause  of  it. 
Left  lung:  In  the  lower  part  of  the  upper  lobe  is  an  area  of  consolidation, 
gray  in  color  and  partly  resolved.  Right  lung:  There  are  a  few  recent 
pleuritic  adhesions.  The  lower  lobe  is  thickly  studded  with  miliary 
tubercles,  and  scattered  through  the  entire  lung  are  suppurating  foci. 
Liver:  There  is  nothing  abnormal.  The  spleen  is  normal.  Pancreas: 
In  and  immediately  around  the  gland  there  are  many  small  abscesses. 
Kidneys :  The  description  of  one  applies  to  both.  The  surface  is  every- 
where and  evenly  dotted  with  minute  white  spots,  which  suggest  septic 
emboli  rather  than  tubercles.  A  few  prominent  white  nodules,  from 

FIG.  132  FIG.  133 


Streptothrix  from  bouillon  culture.  Young  streptothrix  threads  showing 

(From  Tuttle.)  terminal  buds.    (From  Tuttle.) 

one-quarter  inch  to  one-half  inch  in  diameter,  contain  thick,  tenacious 
matter  (Fig.  134).  Section  shows  that  the  entire  substance  of  the 
kidney  is  densely  studded  with  these  minute  white  granules. 

The  gross  pathological  conditions  were  interpreted  as  follows:  An 
old  tuberculous  nodule  in  the  right  lung;  acute  miliary  tuberculosis  of 
the  right  lung  and  peritoneum;  acute  lobar  pneumonia,  affecting  the 
left  lung;  septic  infarctions  and  pysemic  abscesses  of  both  lungs,  heart 
muscle,  both  kidneys,  pancreas,  mesenteric  lymph  nodes,  and  subcu- 
taneous connective  tissue.  The  miliary  tubercles  of  the  right  lung 
and  peritoneum  presented  the  characteristic  appearance  of  genuine 
tuberculosis.  They  were  minute,  hard,  gray,  almost  translucent  nodules, 
while  the  granules  in  the  kidneys  were  of  an  opaque- white  or  yellowish- 
white  color. 

Microscopic  Examination. — Smears  from  the  abscesses  beneath  the 
skin  and  on  the  surface  of  the  kidneys  were  stained  with  methyl-blue, 
carbol-fuchsin,  and  by  Gram's  method.  The  smears  resemble  those 
made  of  tenacious  sputum.  There  is  a  large  amount  of  mucoid  material 


MICRO-ORGANISMS  BELONGING  TO  THE  HIGHER  BACTERIA     427 

containing  a  considerable  number  of  leukocytes.  Occasionally  irreg- 
ularly curved,  thread-shaped  micro-organisms  are  found.  They  vary 
considerably  in  length  and  thickness,  and  broken  and  apparently 
degenerating  fragments  are  seen.  The  more  slender  threads  are  evenly 
stained,  but  some  fragmentation  or  beading  of  the  protoplasm  can 
generally  be  observed.  The  thicker  threads  and  broken  fragments  show 
deeply  stained  globules  and  irregular  bodies  in  a  faintly  visible  rod 
or  thread-shaped  covering.  Some  branching  threads  are  observed, 
but  more  commonly  they  are  not  branching.  The  fragments  seen  in 
the  smears,  varying  in  length  and  thickness  and  staining  properties, 
convey  the  impression  of  a  branching  organism;  the  slender,  more 
evenly  stained  threads  being  the  younger  branches,  and  the  thicker, 
broken,  and  granular  fragments  the  parent  trunks.  No  other  micro- 
organisms are  found  in  the  smears.  Sections  from  the  lower  lobe  of 


Fio.  134 


Portion  of  kidney  showing  minute  and  large  areas  of  infection. 

the  right  lung,  stained  with  haematoxylin  and  eosin,  showed  in  certain 
places  the  identical  microscopic  appearances  which  are  considered 
characteristic  of  tuberculosis.  There  are  distinct  tubercles,  of  recent 
growth,  consisting  of  epithelioid  cells  and  giant  cells.  Others  have 
granular  or  cheesy  centres  with  epithelioid  cells  and  giant  cells  at  the 
periphery;  and  some  consist  of  cicatricial  connective  tissue  surrounded 
by  a  zone  of  epithelioid  cells  containing  giant  cells.  Many  sections 
from  the  right  lung  showing  the  tuberculous  lesions  were  stained  in 
the  usual  way  for  tubercle  bacilli.  Most  careful  searching  by  a  number 
of  competent  examiners  failed  to  discover  any  tubercle  bacilli  or  any 
other  micro-organisms.  Stained  by  Gram's  method,  with  care  not  to 
decolorize  too  completely,  threads  like  those  described  in  the  abscesses 
are  found  in  great  abundance,  but  rather  faintly  stained.  No  threads 
can  be  found  within  the  typical  tubercles  with  giant  cells,  but  in  the 


428  BACTERIA  PATHOGENIC  TO  MAN 

zones  of  small  cells  around  them  they  are  seen  in  great  numbers,  wind- 
ing about  among  the  cells  and  forming  a  sort  of  network.  In  the  minute 
foci  of  small  cells  one  or  two  fragments  of  threads  are  generally  seen, 
and  a  moderate  number  in  the  small  abscesses.  In  the  areas  of  more 
diffuse  infiltration  these  threads  are  abundant.  No  other  micro-organ- 
isms can  be  found  except  in  the  pneumonic  area  of  the  left  lung,  where 
some  groups  of  cocci  are  seen.  The  thread-like  organisms  are  also 
found  in  this  area  and  in  the  other  foci  scattered  through  the  lung. 

The  staining  methods  described  by  Flexner  were  found  to  give  the 
best  results.  The  most  reliable  and  the  one  requiring  the  least  time 
is  a  modified  Gram's  method.  The  sections  stained  with  aniline- 
gentian  violet  are  dipped  for  a  short  time  in  a  diluted  Gram's  iodine 
solution  and  then  treated  with  aniline  oil  until  sufficient  color  has  been 
removed.  The  aniline  oil  is  then  washed  out  with  xylol,  and  the  section 
is  mounted  in  xylol  balsam.  The  other  method  mentioned  by  Flexner 
was  found  to  be  less  reliable,  but  gave  more  beautiful  results  when 
successful.  The  specimens  are  first  stained  with  dilute  hsematoxylin 
solution  to  bring  out  the  nuclei  of  the  cells  and  then  are  stained  with 
carbol-fuchsin  and  decolorized  with  aniline  oil  as  before.  Long  staining 
with  carbol-fuchsin  and  careful  treatment  with  aniline  oil  are  necessary 
for  success.  Many  times  the  micro-organisms  were  completely  decolor- 
ized by  this  method,  but  when  successful  the  dark-red  threads  winding 
among  the  bluish  nuclei  produced  a  striking  picture. 

Culture  Experiments. — Six  tubes  of  Loeffler  blood  serum  were  inocu- 
lated from  the  kidneys.  The  tubes  were  placed  in  the  incubator.  On 
the  third  day  after  inoculation  minute  white  colonies  appeared  in 
some  of  the  tubes,  and  on  the  fifth  day  all  the  tubes  showed  from  three 
to  ten  or  twelve  similar  colonies  in  each.  The  colonies  increased  in 
size  until  some  of  them  reached  a  diameter  of  one-eighth  of  an  inch, 
but  most  of  them  were  smaller.  The  color,  at  first  white,  changed  to 
yellowish-white  and  then  to  a  decided  pale  yellow.  Having  attained 
a  certain  size  at  the  base,  the  colonies  ceased  to  extend,  but  became 
more  and  more  prominent.  The  growth  was  apparently  more  rapid 
at  the  periphery,  and  the  fully  developed  colony  was  round,  with  convex 
sides  and  with  a  cup-shaped  depression  at  the  top.  The  height  of  the 
colony  was  sometimes  greater  and  sometimes  less  than  the  diameter 
of  the  base.  The  well-developed  colonies  cling  firmly  to  the  surface 
of  the  medium  and  are  not  easily  detached  or  broked  up.  The  growths 
in  all  of  the  tubes  were  absolutely  pure,  and  consisted  of  branching 
threads  like  those  found  in  the  sections.  A  more  minute  description 
of  these  organisms  will  be  given  below. 

Transplants  on  Loeffler  blood  serum  produce  a  pale  sulphur-yellow 
growth,  forming  a  layer  with  a  slightly  irregular  and  wrinkled  surface 
and  prominent  edges  where  the  growth  continues  longest.  The  color 
remains  the  same  until  the  medium  dries,  when  it  becomes  white.  The 
growth  clings  so  tenaciously  to  the  surface  of  the  medium  that  in  remov- 
ing a  specimen  for  examination  a  portion  of  the  blood  serum  also  is 
torn  away. 


MICRO-ORGAXIXMS  BELONGING  TO  THE  HIGHER  BACTERIA     429 

Loeffler  blood  serum  seems  to  be  the  most  suitable  medium  for  cul- 
tures. The  growth  on  this  medium  is  more  rapid  and  abundant  than 
on  any  of  the  other  media  tried. 

On  plain  agar  and  glycerin  agar  the  growth  is  the  same  as  on  blood 
serum,  but  is  less  rapidly  developed. 

In  bouillon  the  growth  is  slow  and  takes  place  only  at  the  surface 
and  on  the  sides  of  the  tube.  The  bouillon  remains  perfectly  clear 
and  no  pellicle  or  scum  develops  on  the  surface.  If  the  tube  is  not 
disturbed  or  jarred,  minute  white  tufts  are  seen  clinging  to  the  surface 
of  the  glass.  But  if  the  tube  is  shaken  even  slightly  they  sink  slowly  to 
the  bottom,  forming  a  white,  fluffy  layer.  These  growths  when  undis- 
turbed resemble  minute  balls  of  thistle-down.  The  yellow  color  is  not 
apparent  even  in  the  mass  at  the  bottom  of  the  tube. 

It  is  strictly  aerobic.  In  sealed  tubes  a  very  scant  growth  is  obtained, 
but  when  deprived  of  oxygen  absolutely  no  growth  can  be  detected, 
although  life  is  preserved  for  long  periods.  Cultures  from  a  tube  kept 
for  two  months  without  oxygen,  and  showing  no  sign  of  growth,  devel- 
oped rapidly  when  exposed  to  the  air. 

Morphology. — The  relative  thickness  of  the  threads  varies  some- 
what in  different  parts  of  the  same  individual  branching  organism,  and 
considerably  in  specimens  taken  from  different  culture  media.  If  the 
growth  is  rapid  and  luxuriant,  the  threads  are  thicker  than  in  specimens 
taken  from  growths  on  less  suitable  media.  For  instance,  when  grown 
on  blood  serum  the  threads  are  comparatively  thick  and  coarse,  but 
those  growing  in  bouillon  are  very  slender  and  delicate.  The  main 
trunk  also  is  often  thicker  than  the  branches.  When  unstained  they 
are  homogeneous  gray  threads,  without  any  appearance  of  a  central 
canal  or  double-contoured  wall.  There  is  never  any  segmentation  of 
the  threads.  When  properly  stained  there  is  always  a  distinct  beading 
or  fragmentation  of  the  protoplasm,  but  overstating  with  fuchsin 
produces  rather  coarse,  evenly  red  rods.  The  branching  is  irregular 
and  without  symmetry,  and  the  branches  are  placed  at  a  wide  angle, 
very  nearly,  and  sometimes  quite,  at  right  angles.  This  is  best  seen 
in  specimens  taken  from  liquid  media.  The  irregularly  stellate  arrange- 
ment of  the  branches,  which  was  observed  by  Eppinger  in  his  original 
specimen,  is  often  seen  in  young  organisms  floated  out  from  a  liquid 
medium.  Eppinger  considered  this  form  sufficiently  characteristic  to 
warrant  the  term  asteroides  to  distinguish  the  species.  But  terms  like 
asteroides  and  arborescens  do  not  sufficiently  distinguish  the  species 
of  a  genus  which  is  characterized  by  more  or  less  tree-like  branching, 
and  so  far  no  satisfactory  nomenclature  has  been  adopted. 

Spore  Formation. — On  examining  the  deep-orange  or  red-colored 
growth  upon  potato,  one  is  surprised  to  find  that  the  threads  have 
entirely  disappeared,  and  that  the  specimen  consists  of  moderately 
large  cocci.  In  very  young  cultures  upon  potato  both  threads  and 
cocci  are  found,  but  the  relation  between  them  cannot  be  seen  in  smears 
prepared  in  the  ordinary  way.  In  older  cultures  no  trace  of  the  thread 
can  be  found,  unless  some  fine  granular  matter  may  represent  them 


430  BACTERIA  PATHOGENIC  TO  MAN 

These  cocci  represent  the  spore  form  of  the  organism,  and  when  planted 
upon  blood  serum  the  branching  threads  again  appear.  The  spores 
stain  readily  with  carbol-fuchsin  and  are  not  easily  decolorized.  They 
are  spherical,  or  nearly  so,  but  often  appear  somewhat  elongated, 
apparently  from  beginning  germination.  They  are  killed  by  exposure 
to  moist  heat  of  65°  to  70°  C.  for  an  hour,  but  are  more  resistant  to  dry 
heat.  Exposure  upon  a  silk  thread,  to  a  temperature  above  120°  C. 
for  an  hour  and  a  half  did  not  affect  their  vitality.  The  last  sixty  minutes 
of  this  time  was  at  125°  to  127°  C.,  and  the  bulb  of  the  thermometer 
was  within  the  tube  in  contact  with  the  thread.  No  growth  was  obtained 
after  an  hour's  exposure  at  135°  C.  Drying  destroys  the  threads  after 
a  comparatively  short  time,  but  the  spores  retain  their  vitality  for  an 
indefinite  period.  A  dried-up  potato  culture  which  was  planted  March 
1,  1900,  and  has  stood  in  the  laboratory  ever  since,  still  retains  its 
vitality  at  the  end  of  almost  four  years.  Plants  made  from  this  specimen 
upon  blood  serum  show  an  abundant  yellowish  growth  at  the  end  of 
twenty-four  hours. 

The  germination  of  the  spores  and  the  growth  of  the  threads  were 
observed  under  the  microscope  in  a  hanging  drop  in  the  chamber  of  a 
hollow  slide.  Here  all  stages  of  growth  may  be  seen,  from  the  elongated 
spore  to  the  fully  developed  branching  organism.  The  spore  sends  out 
a  delicate  sprout  which  very  early  looks  like  a  bacillus  with  a  bulbous 
extremity.  The  sprout  soon  branches,  and  the  growth  proceeds  rapidly 
with  frequent  branching  in  all  directions.  Slight  clubbing  of  the  ends 
of  the  branches  is  often  seen,  but  generally  the  ends  are  not  club-shaped. 
In  a  few  stained  specimens  an  extremely  delicate,  almost  colorless, 
club-shaped  structure  was  seen  at  the  ends  of  the  branches.  This 
appearance  was  not  constant,  and  possibly  was  produced  artificially. 
It  can  be  seen  in  some  of  the  photographs. 

The  development  of  the  spores  was  not  so  easily  demonstrated.  In 
specimens  taken  at  frequent  intervals  from  potato  cultures  the  threads 
become  more  and  more  broken  up  into  short  fragments,  and  the  number 
of  free  spores  increases  very  rapidly.  Some  of  the  fragments  appear  to 
have  a  spore  at  each  end,  and  some  have  three  or  more  spores.  The 
free  spores  show  no  regular  arrangement,  but  occasionally  a  short  chain 
of  five  or  six  can  be  found. 

The  identity  of  this  micro-organism  is  not  fully  established.  It  is 
undoubtedly  a  streptothrix,  but  it  does  not  agree  in  all  particulars 
with  any  of  the  varieties  described.  It  approaches  most  nearly  Eppin- 
ger's  variety,  but  at  no  stage  of  its  growth  is  it  ever  motile.  The  color 
and  character  of  the  growth  upon  different  media  and  the  sporulations 
upon  potato  agree  with  the  descriptions  of  the  streptothrix  Eppingeri. 

Animal  Inoculations. — A  number  of  rabbits  and  guinea-pigs  were 
inoculated  subcutaneously  upon  the  abdomen  and  in  the  neighborhood 
of  the  cervical,  axillary,  and  inguinal  lymph  nodes  with  colonies  broken 
up  in  salt  solution.  Indurated  swellings  were  produced  at  the  point 
of  inoculation  and  a  number  of  abscesses  resulted.  The  abscesses 
developed  rapidly  and  some  of  them  opened  spontaneously,  while 


MICRO-ORGANISMS  BELONGING  TO  THE  HIGHER  BACTERIA     431 

others  were  incised.  The  material  evacuated  did  not  resemble  ordinary 
pus,  but  was  thick  and  mucilaginous  and  exceedingly  tenacious,  like 
that  from  the  subcutaneous  abscesses  of  the  patient  described  above. 
The  microscopic  appearance  was  the  same,  and  the  streptothrix 
threads  were  found  in  considerable  numbers.  Pure  cultures  of  the 
streptothrix  were  easily  obtained  from  the  pus  whether  the  abscesses 
ruptured  spontaneously  or  were  incised.  Several  rabbits  and  guinea- 
pigs  and  two  cats  received  peritoneal  inoculations,  but  none  of  them 
showed  any  sign  of  infection.  Sometimes  at  the  point  of  inoculation 
a  few  tuberculous  nodules  wrere  found  at  autopsy,  but  cultures  were 
not  obtained  from  them.  No  local  infection  of  any  consequence  and 
no  general  infection  was  produced  in  this  way.  Thus  far  little  viru- 
lence had  been  shown  by  the  streptothrix  in  inoculation  experiments; 
but  when  rabbits  were  inoculated  intravenously,  a  rapidly  fatal  general 
infection  was  produced,  and  the  lesions  were  similar  in  kind  and 
distribution  to  those  described  in  the  human  subject.  Other  cases 
reported  are  the  following: 

Ferri  and  Faguet  found  in  Bordeaux,  in  a  cerebral  abscess  in  the 
centrum  ovale,  a  branching  fungus,  colored  by  Gram,  which  corre- 
sponded to  the  streptothrix.  It  grew  on  agar  in  round,  ochre-colored 
colonies ;  on  potato  there  was  little  growth  visible ;  slimy,  tough  colonies, 
which  became  gray  and  remained  free  from  white  dusting  on  the  surface. 
Inoculations  in  rabbits  and  guinea-pigs  were  negative. 

CLADOTHRIX  AND  STREPTOTHRIX  IN  CASES  SIMULATING  ACTING- 
MYCOSIS  OR  TUBERCULOSIS.  INTERMEDIATE  CASES  BETWEEN  STREP- 
TOTHRIX AND  ACTINOMYCOSIS. — Gasten  found  in  a  case  of  apparently 
typical  actinomycosis,  in  which  abscess  cavities  were  found  along  the 
spinal  column,  not  the  usual  actinomyces  in  the  yellow,  granular  pus, 
but  a  fine  mass  of  filament.  Cultures  grew  on  all  the  ordinary  media, 
best  at  incubator  temperature,  but  also  at  lower  temperature  on  gelatin. 
The  gelatin  stick  culture,  which  was  especially  characteristic,  formed 
on  the  surface  a  whitish  button;  delicate  thread  stretched  out  in  all 
directions  from  the  point  of  inoculation.  On  agar  and  potato  rumpled, 
folded  films  with  white  deposit  on  the  surface,  which  contained  spores. 
Animal  inoculation  gave  positive  results  only  in  a  few  cases  of  intra- 
peritoneal  injection  of  rabbits  and  guinea-pigs.  Purulent  nodules  were 
found  in  the  peritoneum.  Gasten  called  the  organism  "cladothrix 
liquefaciens." 

Sabraces  and  Riviere  found,  in  a  case  of  cerebral  abscess  and  a  case 
of  chronic  lung  disease  with  occurrence  of  subacute  abscesses,  fungi 
which  differed  from  actinomyces.  The  organisms  were  contained  in 
the  lungs  and  pus  in  the  latter  in  pure  culture.  They  grew  best  at  37° 
C.  in  the  presence  of  oxygen.  On  agar  plates  round,  wart-like  colonies 
were  found  with  yellowish  under  and  whitish  upper  surface.  Grew 
particularly  well  on  fat  and  glycerin  media;  in  milk  a  flesh-colored  rim 
was  developed;  in  glycerin  agar  a  rough,  brownish  deposit,  becoming 
black  with  age.  Gelatin  was  liquefied.  The  culture  had  a  strong  odor 
of  old  mould.  A  yellowish  pigment  was  usually  produced  which  dis- 


432  BACTERIA  PATHOGENIC  TO  MAN 

solved  in  ether.  In  an  atmosphere  of  pure  oxygen  a  brown  pigment. 
Animal  experiments  gave  positive  results  only  when  to  a  fourteen-day- 
old  bouillon  culture  lactic  acid  was  added;  then  pseudotuberculosis 
was  produced. 

Eppinger  found  in  post-mortem  examination  of  a  case  of  chronic 
cerebral  abscess,  which  was  the  result  of  purulent  meningitis,  in  the 
pus  and  abscess  walls,  etc.,  a  delicate  fungoid  growth  which  he  suc- 
ceeded in  cultivating  on  various  media.  On  sugar  agar  it  formed 
yellow,  rumpled  colonies  which  finally  developed  into  a  skin.  On 
potato  it  grew  rapidly,  but  the  colonies  remained  small,  at  first  a  white, 
granular  deposit,  which  afterward  turned  red,  and  on  the  twentieth  day 
resembled  a  crystallized  almond.  It  did  not  grow  well  on  gelatin.  In 
bouillon  it  formed  on  the  surface  a  small  white  granule,  which  became 
deeper  in  the  centre  as  it  grew  and  sunk  to  the  bottom  as  a  white  deposit. 
The  bouillon  remained  clear. 

Microscopically  the  fungus  consisted  of  fine  threads  without  branches, 
which  exhibited  distinct  motility.  No  flagella  were  observed.  It  was 
judged  to  be  a  cladothrix,  to  which  the  name  "asteroides"  was  given 
by  the  author.  It  proved  to  be  quite  pathogenic  for  rabbits  and 
guinea-pigs,  and  produced  an  infection  of  pseudotuberculosis.  Mice 
were  not  affected  by  inoculation. 

Numerous  cases  have  since  been  observed  in  which  the  streptothrix 
proved  to  be  the  cause  of  chronic  lung  diseases,  clinically  suspected  to 
be  tuberculosis. 


CHAPTER  XXXIV. 

THE   PATHOGENIC  FUNGI  AND  YEASTS  (BLASTOMYCETES)— DIS- 
EASES DUE  TO  MICRO-ORGANISMS  NOT  YET  IDENTIFIED. 

The  Fungi. 

MOST  of  the  fungi  are  not  pathogenic  and  interest  us  merely  as  organ- 
isms which  are  apt  to  infect  our  bacteriological  media.  Some  are, 
however,  true  parasites,  and  already  we  know  that  ringworm,  favus, 
thrush,  and  pityriasis  versicolor  are  caused  by  fungi.  Only  those  causing 
ringworm,  favus,  pityriasis,  and  soor  will  be  touched  on. 

Trichophyton  (Ringworm  Fungus). 

Ringworm  of  the  body  or  hairless  parts  of  the  skin,  tinea  circinata, 
and  ringworm  of  the  hairy  parts,  tinea  tonsurans  and  tinea  barbce  or 
tinea  sycosis,  are  due  to  the  fungus  trichophyton,  discovered  by  Gruby 
in  the  human  hair,  and  between  the  epidermal  cells  by  Hebra,  and 
obtained  in  free  cultures  by  gravity. 


FIG.  135 


Hair  riddled  with  ringworm  fungus.    Megalosporon  variety. 

According  to  Sabouraud,  whose  conclusions  are  based  on  an  exten- 
sive series  of  microscopic  examinations  of  cases  of  tinea  in  man  and 
animals,  of  cultivation  in  artificial  media,  and  of  inoculation  on  man 
and  animals,  there  are  two  distinct  types  of  the  fungus  trichophyton 
causing  ringworm  in  man — one  with  small  spores  (2  to  3  mm.)  which 
he  calls  "T.  microsporon,"  and  one  with  large  spores  (7  to  8  mm.) 
which  he  calls  "T.  megalosporon."  They  differ  in  their  mode  of  growth 

28 


434 


BACTERIA  PATHOGENIC  TO  MAN 


on  artificial  media  and  in  their  pathological  effects  on  the  human  skin 
and  its  appendages.  T.  microsporon  is  the  common  fungus  of  tinea 
tonsurans  of  children,  especially  of  those  cases  which  are  rebellious 
to  treatment,  and  its  special  seat  of  growth  is  in  the  substance  of  the 
hair.  T.  megalosporon  (Fig.  135)  is  essentially  the  fungus  of  ringworm 
of  the  beard  and  of  the  smooth  part  of  the  skin;  the  prognosis  as  regards 
treatment  is  good.  One-third  of  the  cases  of  T.  tonsurans  of  children 
are  due  to  trichophyton  megalosporon.  The  spores  of  T.  microsporon 
are  contained  in  a  mycelium;  but  this  is  not  visible,  the  spores  appearing 


FIG.  136 


These  two  half-plates  show  three  months'  growth  on  peptone-maltose  agar  of  two  megalosporon 
varieties  of  the  ringworm  fungus.    Natural  size. 

irregularly  piled  up  like  zooglosa  masses;  and,  growing  outside,  they 
form  a  dense  sheath  around  the  hair.  The  spores  of  T.  megalosporon 
are  always  contained  in  distinct  mycelium  filaments,  which  may  either 
be  resistant  when  the  hair  is  broken  up  or  fragile  and  easily  breaking 
up  into  spores.  The  two  types  when  grown  in  artificial  cultures  show 
distinct  and  constant  characters.  The  cultures  of  T.  microsporon  show 
a  downy  surface  and  white  color;  those  of  T.  megalosporon  a  powdery 
surface,  with  arborescent  peripheral  rays,  and  often  a  yellowish  color. 
Although  the  morphological  appearances,  mode  of  growth,  and  clinical 
effects  of  each  type  of  trichophyton  show  certain  characters  in  general, 


THE  PATHOGENIC  FUNGI  AND  YEASTS  435 

yet  there  are  certain  constant  minor  differences  which  point  to  the  fact 
that  there  are  several  different  kinds  of  species  of  fungus  included  under 
t'jirli  type.  The  species  included  under  T.  microsporon  are  few  in 
number,  and,  with  the  exception  of  one  which  causes  the  common  con- 
tagious "herpes"  of  the  horse,  almost  entirely  human.  The  species  of 
T.  megalosporon  are  numerous  and  fall  under  several  natural  groups, 
the  members  of  which  resemble  one  another  both  from  clinical  and 
mycological  aspects  (Fig.  136).  Many  animals  are  subject  to  the  growth 
upon  their  skins  of  particular  species  of  T.  megalosporon. 

Achorion  Schoenleinii  (Favus). 

Favus  is  due  to  a  fungus  discovered  by  Schoenlein  in  1839,  and  called 
by  Remak  Achorion  schoenleinii.  The  disease  is  communicated  by 
contagion,  the  fungus  being  often  derived  from  animals,  especially 
cats,  mice,  rabbits,  fowls;  and  dogs  are  also  subject  to  it.  It  grows 
much  more  slowly  than  the  ringworm  fungus,  and  is,  therefore,  not  so 

FIG.  137 


A  portion  of  a  favus-infected  hair  ;  magnified. 


easily  transmitted.  Want  of  cleanliness  is  a  predisposing  factor.  The 
fungus  seems  to  find  a  more  favorable  soil  for  its  development  on  the 
skin  of  persons  in  weak  health,  especially  from  phthisis,  than  in  others. 
Pathologically,  the  disease  represents  the  reaction  of  the  tissues  to 
the  irritation  caused  by  the  growth  of  the  fungus.  The  spores  generally 
find  their  way  into  the  hair  follicles,  where  they  grow  in  and  about  the 
hair  (Fig.  137).  The  favus  fungus  grows  in  the  epidermis,  the  density 
of  the  growth  causing  pressure  on  the  parts  below,  thus  crushing  out 
the  vitality  of  the  hair  and  giving  rise  to  atrophic  scarring.  The  dis- 
ease shows  a  marked  preference  for  the  scalp,  but  no  part  of  the  skin 
is  exempt,  and  even  the  mucous  membranes  are  liable  to  be  attacked. 
Kaposi  has  reported  a  case  in  which  a  patient  suffering  from  universal 
favus  died,  with  symptoms  of  severe  gastrointestinal  irritation,  which  was 
found  after  death  to  be  due  to  the  presence  of  the  favus  fungus  in  the 
stomach  and  intestine.  On  the  scalp  it  first  appears  as  a  tiny  sulphur- 
yellow  disk  or  scidulum,  depressed  in  the  centre  like  a  cup  and  pierced 
bv  a  hair.  This  is  the  characteristic  lesion.  The  cup  shape  is  attributed 


436  BACTERIA  PATHOGENIC  TO  MAN 

by  Unna  to'growtr/ atjlie  sides  proceeding  more  vigorously  than  at  the 

centre. 
There  is  some  difference  of  opinion  as  to  whether  there  is  only  one 

or  several  varieties  of  favus  fungus.    It  was  suggested  by  Quincke  that 

there  are  three  different  species  of  favus 
fungus.  Later  investigations  have  appa- 
rently shown,  however,  that  the  achorion 
Schoenleinii  is  the  only  fungus  of  favus. 

The  favus  fungus  is  readily  cultivated  at 
the  body  temperature,  and  also  at  room 
temperature,  in  the  ordinary  culture  media, 
as  agar,  blood  serum,  gelatin,  bouillon, 
milk,  infusion  of  malt,  eggs,  potato,  etc. 
(Fig.  138).  The  growth  develops  slowly 
and  shows  a  preference  to  grow  beneath 
the  surface  of  the  medium — except  on 
potato,  upon  which  it  develops  on  the  sur- 

Five-months-old  colony  of  favus  on     ^  -,  L  rp,,          ,  .   ,.     £  « 

peptone-maltose  agar;  actual  size.      face  m  layers.     Ihe  characteristic  torm  ot 

growth  is    that   of    moss-like    projections 

from  a  central  body.  The  color  is  at  first  grayish- white,  then  yellowish. 
As  seen  under  the  microscope,  ray-like  mycelium  filaments  are  devel- 
oped, which  divide  into  branches.  The  ends  are  often  swollen  or 
club-shaped,  and  there  are  various  enlargements  along  the  body  of 
the  filament. 

Pityriasis  Versicolor. 

This  organism  belongs  to  a  group  of  fungi  which,  in  contrast  to  the 
more  parasitic  fungi,  favus  and  trichophyton,  invades  only  the  most 
superficial  layers  of  the  skin.  It  does  not  penetrate  the  deeper  layers 
nor  does  it  give  rise  to  any  considerable  pathological  changes  in  the 
skin  or  hair.  Although  the  vegetative  elements  of  these  fungi  are  much 
more  numerous  in  the  affected  portions  of  skin  than  is  the  case  with 
the  more  parasitic  species,  they  are  not  nearly  as  contagious  as  the 
latter. 

By  preference  pityriasis  versicolor  attacks  the  chest,  abdomen,  back, 
and  axillse;  less  frequently  neck  and  arms,  while  exceptionally  it  attacks 
also  the  face.  The  growth  shows  itself  as  scattered  spots  varying  in 
color  from  that  of  cream-coffee  to  reddish-brown.  The  spots  are 
readily  scraped  off  and  show  fine  lamellation  or  scaling.  Occasionally 
the  spots  are  confluent,  and  sometimes  arranged  in  ring  form  like  herpes 
tonsurans. 

In  spite  of  their  slight  contagiousness  this  is  one  of  the  most  frequent 
dermatomycoses.  Although  it  is  distributed  widely  over  the  earth,  it 
is  more  frequently  observed  in  southern  than  in  northern  countries. 

Persons  with  a  tender  skin  and  a  disposition  to  perspire  freely  are 
particularly  affected  by  pityriasis  versicolor,  and  this  is  undoubtedly  the 
only  reason  why  the  affection  is  so  frequently  observed  in  consumptives. 


THE  PATHOGENIC  FUNGI  AND  YEASTS  437 

Women  are  more  frequently  attacked  than  men,  while  children  and 
old  people  are  rarely  affected. 

The  source  of  infection  is  unknown,  since  the  absence  of  contagion 
has  frequently  been  demonstrated.  It  seems  likely  that  the  spores  of 
this  fungus  are  so  widely  distributed  that  susceptible  individuals  are 
easily  infected. 

The  arrangement  of  the  fungus  in  the  scales  of  epidermis  is  char- 
acteristic. The  short  and  thick-curved  hyphse  (7/>«  to  13/>«  long  and  3/* 
to  4/*  wide)  surround  large  clumps  of  spores.  The  spores  are  coarse, 
doubly  contorted  (4//  to  7/*)  or  round.  On  staining  with  Ziehl's  solution 
the  spores  are  seen  to  contain  deeply  stained  globules  lying,  in  all  prob- 
ability, on  the  inner  surface  of  the  cell  membrane.  The  rest  of  the 
protoplasm  is  but  little  stained,  or  not  at  all.  One  frequently  finds 
that  these  globules  have  disintegrated  into  numerous  fine  granules. 
The  globules  are  also  found  free;  what  their  nature  is  does  not  appear; 
they  are  not  found  in  cultures,  the  freshly  developed  spores  showing 
only  a  single  globular  mass  of  protoplasm  possessing  a  fine  blue  lustre. 

Soor  Fungus  (Thrush). 

Soor,  as  is  well  known,  occurs  most  frequently  in  the  oral  mucous 
membrane  of  infants  during  the  early  weeks  of  life.  It  is  also  found 
as  a  slight  mycosis  in  the  vagina,  especially  of  pregnant  women.  In 
rare  cases  the  disease  attacks  adults,  and  then  especially  those  whose 
system  has  beeji  undermined  by  other  diseases,  such  as  diabetes, 
typhoid  patients,  etc.  A  few  cases  are  recorded  in  the  literature  in 
which  this  fungus  has  given  rise  to  constitutional  disease.  In  these 
cases  autopsy  has  shown  abscesses  in  various  parts  of  the  body,  such 
as  in  the  lungs,  spleen,  kidney,  and  brain. 

In  the  lesions  of  the  disease  as  well  as  in  cultures,  this  fungus  appears 
both  as  an  yeast  and  a  mycelium.  The  yeast  cells  are  oval  in  form, 
about  5,u  to  6,u  long  and  4/*  wide,  and  can  in  no  way  be  distinguished 
from  other  yeast  cells  either  by  their  appearance  or  their  method  of 
propagation.  The  threads  of  the  mycelium  vary  very  much  in  length 
and  thickness,  and  show  all  intermediate  forms  between  a  typical  and 
a  budding  mycelium. 

Soor  is  not  much  influenced  by  acids  or  alkalies,  growing  well  both 
in  acid  and  in  alkaline  media.  On  the  other  hand,  it  is  very  susceptible 
to  the  common  disinfectants,  especially  salicylic  acid,  corrosive  sub- 
limate, phenol,  etc.  This  fact  is  made  use  of  in  local  treatment. 


Blastomycetes    Yeasts; . 

These  micro-organisms  are  of  the  greatest  importance  in  brewing 
and  baking,  but  as  yet  no  important  pathological  lesions  in  man  have 
been  attributed  to  them,  although  certain  recent  experiments  have 
shown  that  some  varieties  when  injected  are  capable  of  producing 
tumors  and  many  are  pathogenic  for  mice.  They  are  not  uncommonly 


438  BACTERIA  PATHOGENIC  TO  MAN 

present  in  the  air  and  in  cultures  made  from  the  throat.  They  consist 
of  round  or  oval  cells,  usually  many  times  larger  than  the  bacteria. 
They  usually  reproduce  themselves  by  budding,  a  portion  of  the  proto- 
plasm budding  and  finally  being  cut  off  to  form  a  new  individual. 

For  many  centuries  blastomycetes,  or  yeasts,  have  proven  themselves 
to  be  of  great  benefit  to  man,  untold  millions  of  them  being  used  daily 
in  breweries,  distilleries,  and  other  industries.  Until  a  few  years  ago 
this  group  of  organisms  stood  alone  among  other  allied  forms  of  life 
as  being  the  only  one  in  which  pathogenic  species  were  unknown.  It 
is  not  more  than  ten  years  since  the  discovery  of  the  first  of  the  disease- 
producing  yeasts.  Since  that  time  these  organisms  have  been  studied 
not  only  because  of  their  interesting  biological  and  physiological 
characteristics,  but  also  from  the  point  of  view  of  the  physician  and 
etiologist.  Our  present  knowledge  concerning  the  pathogenic  yeasts 
may  be  briefly  summarized  as  follows: 

The  position  which  the  yeasts  occupy  in  systematic  biology  (botany) 
has  not,  thus  far,  been  accurately  determined.  In  fact,  it  is  even  doubted 
whether  they  constitute  independent  fungi  or  are  perhaps  a  particular 
form  of  growth  of  more  highly  organized  fungi,  especially  of  the  mould 
fungi.  This  hypothesis  was  formulated  by  Brefeld  about  thirty  years 
ago,  but  has  not  thus  far  been  proved.  For  the  present  it  seems  advis- 
able to  retain  the  yeasts  in  a  group  of  fungi  by  themselves. 

The  chief  characteristic  of  the  yeasts  is  their  peculiar  method  of 
reproduction  which  in  most  cases  is  by  means  of  budding.  For  this 
reason  these  organisms  go  by  the  name  of  blastomycetes  in  contrast  to 
the  fission  fungi,  or  schizomycetes,  and  the  mould  fungi,  or  hyphomycetes. 
A  transition  between  the  blastomycetes  and  the  hyphomycetes  is 
formed  by  the  oidien,  which  at  one  time  grow  to  long  threads,  at  another 
time  (under  certain  conditions  almost  exclusively)  multiply  by  budding. 
But  no  hard-and-fast  line  exists  between  these  classes,  for  the  yeasts 
can  at  times  develop  short  hyphoe,  at  other  times,  in  rare  cases,  form 
new  individuals  by  segmentation. 

The  most  important  property  of  yeasts,  though  one  not  possessed 
by  all  to  the  same  degree,  is  that  of  producing  alcoholic  fermentation. 
In  practice  we  distinguish  between  the  yeasts  that  can  be  employed 
practically,  "culture  yeasts,"  and  those  which  often  act  as  disturbing 
factors,  so-called  "wild"  yeasts. 

The  shape  of  most  of  the  culture  yeasts  is  oval  or  elliptical  (Fig.  139). 
Round  or  globular  forms  are  more  often  met  with  among  the  wild 
species  and  such  as  excite  only  a  slight  degree  of  fermentation.  They 
are  known  as  "  torula  "  forms.  But  sausage-shaped  and  thread  forms 
are  also  met  with. 

The  individual  yeast  cells  are  strongly  refractive,  so  that  under  the 
microscope  at  times  they  have  almost  the  lustre  of  fat  droplets.  This 
is  important  because  in  examining  fresh  tissues  the  yeast  cells  may  be 
hard  to  distinguish  from  fat  droplets,  often  requiring  the  aid  of  certain 
reagents  for  their  identification. 

The  size  of  the  individual  yeast  cells  varies  enormously,  even  in 


THE  PATHOGENIC  FUNGI  AND  YEASTS  439 

those  of  the  same  species  or  the  same  culture.  In  old  colonies  indi- 
viduals may  be  found  hardly  larger  than  cocci,  1  to  2/t,  while  in  other 
colonies,  especially  on  the  surface  of  a  liquefied  medium,  giant  yeast 
cells  are  found  often  attaining  a  diameter  of  40//  or  more.  In  spite  of 
these  wide  fluctuations,  however,  the  various  species  are  characterized 
by  a  fairly  definite  average  in  size  and  form. 

During  the  process  of  budding  the  nucleus  of  the  cell  moves  toward 
the  margin,  where  it  divides.  At  this  point  the  limiting  membrane  of 
the  cell  ruptures  or  a  hernia-like  protrusion  develops  which  has  the 
appearance  of  a  button  attached  to  the  cell.  The  daughter-cell  so 
formed  rapidly  increases  in  size  and  gradually  assumes  the  shape  and 
size  of  the  mother-cell. 

A  fact  of  the  utmost  importance  for  the  propagation  of  the  blasto- 
mycetes  and  continuation  of  the  species  is  the  formation  of  spores.  In 
this  also  the  cell  nucleus  takes  part,  dividing  into  several  fragments, 


FIG.  139 


.-•-* 


. 

- 


f 


£„< 


"•••-*  '•• 


Saccharomyces  Busse.    X  350  diameters.    (From  Kolle  and  Wassermann.) 

each  of  which  becomes  tlie  centre  of  a  new  cell  lying  within  the  original 
cell.  These  new  cells  possess  a  firm  membrane,  a  cell  nucleus,  and  a 
little  dry  protoplasm.  The  number  of  spores  developed  in  the  yeast  cells 
varies,  but  is  constant  for  a  given  species.  As  a  rule,  one  cell  does  not 
produce  more  than  four  endogenous  spores,  so-called  astrospores;  but 
species  have  been  observed — e.  g.,  schizosaccharomyces  octosporas  (Beijer- 
inck) — in  which  eight  spores  are  found. 

The  vitality  of  yeasts  is  truly  enormous.  Hansen  as  well  as  Lindner 
were  able  to*  obtain  a  growth  from  cultures  twelve  years  old.  Busse 
succeeded  in  getting  a  luxuriant  growth  from  a  dry  potato  culture 
seven  and  a  half  years  old,  and  almost  as  hard  as  bone. 

As  stated  above,  the  most  important  property  of  yeasts  is  that  of 
producing  alcoholic  fermentation.  While  a  large  number  of  yeasts  are 
merely  able  to  decompose  dextrose  into  alcohol  and  carbon  dioxide, 
there* are  some  which  ferment  cane-sugar,  others  which  invert  and 
ferment  starch;  in  fact,  all  kinds  of  carbohydrates  may  be  decomposed. 


440  BACTERIA  PATHOGENIC  TO  MAN 

As  was  first  shown  by  Buchner,  the  fermentation  is  due  to  enzymes 
produced  by  the  yeast  cells.  These  enzymes  differ  in  the  different 
species,  and  hence  also,  their  action  differs. 

The  pathogenic  blastomycetes  may  be  briefly  summarized  as  fol- 
lows: 

Saccharomyces  Busse,  isolated  in  1894  by  O.  Busse  from  the  tibia  of 
a  thirty-one-year-old  woman,  who  died  thirteen  months  after  the  first 
symptoms  appeared.  The  autopsy  showed  numerous  broken-down 
nodules  on  several  of  the  bones,  in  the  lungs,  spleen,  and  kidneys. 
The  yeast  was  cultivated  from  all  these  foci  (Fig.  139). 

Saccharomyces  subcutaneus  tumefaciens,  isolated  in  1895  by  Curtis. 
The  patient  was  a  young  man  showing  multiple  tumors  on  the  hips 
and  neck  having  the  gross  appearance  of  softened  myxosarcomata. 

This  yeast  is  pathogenic  for  rats,  mice,  and  dogs,  only  slightly  so  for 
rabbits,  and  not  at  all  for  guinea-pigs. 

Gilchrist  has  described  a  case  of  pseudolupus  vulgaris  caused  by  a 
blastomyces.  The  disease  lasted  ten  years,  during  which  time  many 
nodules  developed  on  the  face,  back  of  the  hands,  scrotum,  thigh,  and 
neck.  The  nodules  ulcerated  and  then  healed,  leaving  scars.  Busse 
believes  that  this  organism  should  be  classed  with  oidium,  and  this 
opinion  is  shared  by  Buschke. 

In  a  case  reported  by  Hektoen  in  1899  that  author  describes  skin 
lesions  very  similar  to  those  observed  by  Gilchrist.  The  organism 
obtained  on  cultivation,  however,  differed  somewhat  from  Gilchrist's. 
Injected  into  rats  this  yeast  produced  abscesses  and  caused  the  death 
of  the  animal  in  five  days. 

Lundsgaard  reported  a  case  of  ophthalmia  due  to  an  yeast.  His 
patient,  a  man  thirty-four  years  old,  had  a  severe  hypopyon  keratitis, 
in  the  pus  of  which  many  yeasts  were  present.  Pure  cultures  of  the 
yeast  inoculated  into  guinea-pigs  produced  abscess  both  at  the  site  of 
inoculation  and  in  the  lymph  glands. 

Buschke  isolated  an  yeast  from  a  cervical  discharge  in  which  no 
gonococci  were  present.  The  yeast  was  pathogenic  for  guinea-pigs. 

In  1895  Dr.  G.  Tokishige  reported  that  an  epidemic  quite  common 
among  horses  in  Japan,  known  as  "Japanese  worm/7  "benign  worm," 
or  "pseudoworm,"  is  caused  by  Saccharomyces.  The  disease  begins 
in  the  skin  in  the  form  of  hard,  painless  nodules  from  the  size  of  a  pea 
to  a  walnut.  These  break  down  and  give  rise  to  gradually  extending 
ulcers.  Pure  cultures  of  the  Saccharomyces  are  pathogenic  only  for 
horses,  not  for  rabbits,  guinea-pigs,  or  hogs.  In  the  districts  where 
the  disease  prevails  among  horses  it  is  also  frequently  seen  in  cattle. 

Shortly  after  Tokishige's  publication  a  similar  disease  occurring  in 
horses  in  Italy  and  Southern  France  was  identified  as  being  caused  by 
Saccharomyces.  Cultures  of  this  yeast  however,  differ  somewhat  from 
that  obtained  in  Japan ;  so  that  Busse  is  inclined  to  regard  the  two  as 
two  different  species  of  blastomycetes. 

In  recent  years  the  attempt  has  been  made  to  connect  the  develop- 
ment of  cancerous  growth  with  blastomycetes.  This  is  due  in  a  meas- 


THE  PATHOGENIC  FUNGI  AND  YEASTS  441 

lire  to  a  certain  similarity  between  the  yeasts  and  the  cell  inclusions 
or  so-called  "parasites"  of  cancer,  and,  further,  to  the  fact  that  when 
yeasts  are  injected  into  the  animal  body  tumor-like  nodules  are  often 
developed  at  the  site  of  inoculation  and  in  the  internal  organs.  But 
these  nodules  are  not  tumors  in  the  pathological  sense  of  the  term, 
but  merely  masses  of  blastomycetes  mixed  with  inflammatory  tissue 
proliferations  to  a  very  variable  degree.  At  the  present  time  Sanfelice 
and  his  pupils  are  perhaps  the  only  ones  who  regard  the  thickenings 
produced  in  the  tissues  by  Saccharomyces  neoformans  as  true  tumors. 
His  work,  however,  is  not  at  all  convincing. 

Diseases  in  which  the  Micro-organisms  Exciting  them  are  as 

yet  Undetected. 

Measles. — Many  bacteria  as  well  as  bodies  supposed  to  be  protozoa 
have  been  described  by  various  investigators  as  occurring  on  the  mucous 
membranes  or  in  the  blood  of  those  sick  of  measles.  None  of  these 
have  been  established  as  the  exciting  factor.  Hektoen  has  recently 
transferred  blood  from  a  case  of  measles  to  two  individuals  and  so 
communicated  the  disease. 

Scarlet  Fever. — Both  streptococci  and  protozoa  have  been  described 
as  the  exciting  factors  in  this  disease,  as  already  previously  mentioned. 
The  streptococci  are  certainly  present,  but  are  looked  upon  by  most  as 
secondary  invaders.  They  undoubtedly  add  greatly  to  the  gravity  of 
the  disease.  The  bodies  described  by  Mallory  as  protozoa  are  still 
under  investigation.  Serum  treatment  has  been  used  to  overcome  the 
streptococcus  infection.  The  best  results  have  been  obtained  in  Vienna, 
and  by  Moser.  He  uses  a  serum  obtained  from  horses  receiving  multiple 
cultures  from  cases  of  scarlet  fever.  Only  about  one  horse  in  three  gives 
a  curative  serum.  The  doses  used  are  very  large  (100  to  200  c.c.).  The 
results  claimed  are  very  striking. 

Typhus  Fever. — Nothing  has  as  yet  been  determined  concerning 
the  micro-organisms  exciting  this  disease. 

Smallpox. — Streptococci  as  secondary  invaders  add  here,  as  in  scarlet 
fever,  a  dangerous  infection.  The  status  of  protozoa  is  described  fully 
under  the  section  on  Protozoa. 

Syphilis. — The  bacilli  described  by  Lustgarten  and  others  are  now 
no  longer  considered  as  a  factor.  Schaudinn's  recent  discovery  of 
spirochsete  is  considered  under  the  protozoa. 

Rabies  (Hydrophobia). — No  bacteria  have  been  discovered  that 
are  considered  as  factors.  The  possibility  of  the  negri  bodies  being 
protozoa  and  the  exciting  factor  is  considered  under  Protozoa. 

Whooping-cough. — Jochmann  and  Krause,  in  Germany,  and  Woll- 
stein,  in  this  country,  have  shown  that  bacilli  differing  slightly  in  cul- 
tural reactions  and  in  agglutination  from  typical  influenza  bacilli  can 
be  detected  in  practically  all  cases  of  whooping-cough  during  the  acute 
stages.  Wollstein  proved  that  the  blood  of  cases  of  whooping-cough 
agglutinated  these  bacilli  frequently  in  dilutions  of  1:200  and  over. 


442  BACTERIA  PATHOGENIC  TO  MAN 

The  question  is  still  undecided  as  to  whether  these  bacilli  are  anything 
more  than  very  frequent  causes  of  secondary  infection. 

Pemphigus  Neonatorum. — Several  micrococci  have  been  described 
as  the  cause  of  infection. 

Impetigo  Contagiosa. — The  findings  have  been  similar  to  those  in 
pemphigus. 

Scurvy. — This  disease  is  probably  not  due  to  micro-organisms. 

Mumps. — Diplococci  have  been  considered  by  several  investigators 
as  possibly  being  the  exciting  organisms. 

Noma. — It  is  as  yet  undecided  whether  this  disease  is  due  to  one 
or  to  several  micro-organisms.  A  special  predisposition  of  the  tissues 
is  necessary.  A  streptothrix,  pseudodiphtheria  bacilli,  and  diphtheria 
bacilli  have  been  the  organisms  most  frequently  present. 

Articular  Rheumatism. — The  specific  organisms  of  this  disease 
have  been  sought  in  the  synovial  fluid,  blood,  vegetations  on  heart 
valves,  and  in  exudates  on  tonsils,  etc.  Streptococci  have  been,  of  all 
bacteria,  most  frequently  found.  They  grow  in  short  chains  or  as 
diplococci.  Most  bacteriologists  believe  the  exciting  factor  has  not 
yet  been  identified  and  that  the  streptococci  and  other  bacteria  are 
secondary  infections. 

Beriberi. — Micro-organisms,  both  of  bacterial  and  protozoan 
nature,  have  been  considered  as  the  exciting  factor,  but  nothing  definite 
has  been  proven. 

Yellow  Fever. — The  bacillus  described  by  Sanarelli  as  the  exciting 
factor  is  now  known  to  be  at  most  a  rather  frequent  secondary  invader. 
Reed,  Carroll,  and  Agramonte  have  shown  that  the  stegomyia  fasciata 
is  the  only  carrier  of  the  infecting  agent.  Twelve  days  after  biting  a 
yellow-fever  patient  the  mosquito  is  able  to  infect  a  non-immune  by 
biting.  The  insect  continues  for  a  number  of  weeks  to  be  capable  of 
infecting  man.  The  blood  of  yellow-fever  patients  is  only  capable  of 
infecting  mosquitoes  during  the  first  few  days.  It  has  also  been  im- 
possible to  infect  mosquitoes  by  letting  them  bite  the  body  of  a  person 
who  has  died  of  yellow  fever.  The  blood  of  yellow-fever  patients  in- 
jected into  non-immunes  produced  the  disease.  The  virus  of  yellow  fever 
is  apparently  capable  of  passing  through  a  Berkefeld  filter.  At  present 
nothing  is  known  about  the  micro-organism.  The  bite  of  the  mos- 
quito is  the  only  known  method  of  causing  infection.  The  clothing 
and  the  discharges  from  the  mouth,  kidney,  and  intestines  are  harm- 
less. 

Dengue. — The  organism  exciting  this  disease  is  unknown.  An 
influenza-like  bacillus  has  been  observed  as  frequently  present.  The 
mosquito  has  also  been  suspected  as  the  carrier  of  infection. 

Invisible  Micro-organisms. — There  are  a  number  of  diseases  from 
which  the  infectious  material  passes  through  stone  filters  which  are 
known  not  to  allow  the  passage  of  visible  micro-organisms.  The  horse 
sickness  of  South  Africa,  yellow  fever,  and  the  cattle  plague,  pleuro- 
pneumonia  of  cattle,  are  of  this  nature. 


CHAPTER  XXXV. 

THE    BACTERIOLOGICAL   EXAMINATION  OF  WATER,  AIR,  AND 

SOIL— THE  CONTAMINATION  AND  PURIFICATION  OF 

WATER— THE  DISPOSAL  OF  SEWAGE. 

THE  bacteriological  examination  of  water  is  undertaken  with  the 
purpose  of  discovering  whether  any  pathogenic  bacteria  are  liable 
to  be  present.  The  determination  of  the  number  of  bacteria  in  water 
was  for  a  time  considered  of  great  importance,  then  it  fell  into  disrepute, 
and  the  attempt  was  made  to  isolate  the  specific  germs  of  diseases  which 
were  thought  to  be  water-borne.  At  first  these  attempts  seemed  very 
successful  in  that  supposed  typhoid  bacilli  and  cholera  spirilla  were 
found.  Further  study  revealed  that  there  were  common  water  and 
intestinal  bacteria  which  were  so  closely  allied  to  the  above  forms 
that  the  tests  applied  did  not  separate  them.  Even  the  use  of  a  serum 
from  an  animal  immunized  to  injections  of  the  typhoid  bacillus  was 
found  to  agglutinate  some  other  bacteria  in  high  dilutions;  so  that  the 
test  as  usually  carried  out  was  insufficient.  With  the  latest  technique 
it  is  probable  that  the  serum  from  an  immunized  animal  from  which 
the  group  agglutinins  have  been  absorbed  will  be  sufficient  to  identify 
any  bacillus  which  it  agglutinates.  The  practical  impossibility  of  get- 
ting typhoid  bacilli  from  suspected  water  caused  a  return  to  the  esti- 
mation of  the  number  of  bacteria  in  water  and  above  all  to  the  estima- 
tion of  the  number  of  intestinal  bacteria.  It  is  known  that  the  group 
of  colon  bacilli  have  about  the  same  duration  of  life  as  the  typhoid 
bacilli,  and  as  the  colon  bacilli  come  chiefly  or  wholly  from  the  intes- 
tinal passages  of  men  and  animals,  it  was  fair  to  assume  that  typhoid 
bacilli  could  not  occur  without  the- presence  of  the  colon  bacillus.  The 
latter  could,  of  course,  occur  abundantly  without  the  typhoid  bacillus. 

During  the  past  few  years  the  attention  of  sanitarians  has  peen  seri- 
ously devoted  to  the  interpretation  of  the  presence  of  smaller  or  larger 
numbers  of  colon  bacilli  in  water,  until  at  present  upon  the  quantitative 
analysis  (measuring,  within  certain  limits,  decomposing  organic  matter) 
and  the  colon  test  (indicating  more  specifically  that  derived  from  intes- 
tinal discharges;  the  bacteriological  analysis  of  water  is  based. 

Technique  of  Quantitative  Analysis. — The  utmost  care  is  necessary 
to  get  reliable  results.  A  speck  of  dust,  a  contaminated  dish,  a  delay 
of  a  few  hours,  an  improperly  prepared  ji<rar  or  uvlatin,  a  too  high  or 
too  low  temperature,  may  introduce  an  error  which  would  make  a 
reliable  test  impossible. 

COLLECTION  OF  SAMPLES. — The  small  sample  taken  must  represent 
the  whole  from  which  it  was  drawn.  If  a  brook-water,  it  must  be  taken 
some  distance  from  the  bank;  if  from  a  tap,  the  water  in  the  pipes  must 


444  BACTERIA  PATHOGENIC  TO  MAN 

first  run  off,  for  otherwise  the  effect  of  metallic  substances  will  invalidate 
the  results;  if  from  lake  or  pond,  the  surface  scum  or  bottom  mud 
must  be  avoided,  but  may  be  examined  separately.  The  utensils  by 
which  the  water  is  taken  should  be  of  a  good  quality  of  glass,  clean 
and  sterile.  From  a  brook  the  water  can  be  taken  directly  into  a 
bottle,  the  stopper  being  removed  while  it  fills ;  from  a  river  or  pond  it 
can  be  taken  from  the  bow  of  a  small  boat,  the  bottle  being  held  inverted 
on  a  pole  with  a  clamp  until  it  has  entered  the  water;  from  a  well  a 
special  apparatus  has  been  devised  by  Dr.  A.  C.  Abbott,  where  a  bottle 
with  a  leaded  bottom  is  so  held  that  when  lowered  to  the  proper 
depth  a  jerk  will  remove  the  cork  and  allow  the  bottle  to  fill.  The 
same  device  can  be  rigged  up  readily  by  anyone.  The  sample  of  water 
should  be  tested  as  soon  as  possible,  for  the  bacteria  immediately  begin 
to  increase  or  decrease.  In  small  bottles  removed  from  the  light  preda- 
tory micro-organisms  and  many  bacteria  begin  to  increase,  and  among 
these  are  the  members  of  the  colon  group.  Thus,  the  Franklarids  record 
a  case  in  which  in  a  sample  of  well-water  kept  during  three  days  the 
bacteria  increased  from  7  to  495,000;  while  Jordan  found  that  in  a 
sample  the  bacteria  in  forty-eight  hours  fell  from  535,000  to  54,500. 
In  a  sample  I  took  from  the  Croton  River  the  colon  bacilli  during  twenty- 
four  hours  increased  from  10  to  100  per  c.c.  The  only  safe  way  to 
prevent  this  increase  is  to  plate  and  plant  the  water  in  fermentative 
tubes  within  the  space  of  one  or  two  hours.  It  is  far  better  to  make 
the  cultures  in  the  open  field  or  in  a  house  rather  than  to  wait  six  to 
twelve  hours  for  the  conveniences  and  advantages  of  the  laboratory. 
If  sent  to  the  laboratory,  water  should  be  kept  below  40°  C. 

The  third  matter  of  great  importance  is  the  adding  of  proper  amounts 
of  water  to  the  broth  in  the  fermentation  tubes  and  the  media  for  plating. 
Usually  1  c.c.,  0.1  c.c.,  and  0.01  c.c.  are  added  to  the  fermentation 
tubes  and  to  10  c.c.  of  the  melted  nutrient  agar  or  gelatin.  If 
possible  duplicate  tests  should  always  be  made.  When  it  is  desired 
to  know  whether  colon  bacilli  are  present  in  larger  amounts  than  1  c.c., 
quantities  as  great  as  10  or  100  c.c.  can  be  added  to  bouillon,  and  then 
after  a  few  hours  1  c.c.  added  to  fermentation  tubes.  Less  than  ten 
colonies  and  more  than  two  hundred  on  a  plate  give  inaccurate  counts, 
the  smaller  number  being  too  few  to  judge  an  average  and  the  larger 
number  interfering  with  each  other. 

The  chemical  composition  of  the  medium  on  which  the  bacteria  are 
grown  affects  the  results  of  the  analysis.  Nutrient  1  per  cent,  agar 
gives  slightly  lower  counts  than  gelatin,  but  on  account  of  its  con- 
venience in  summer  and  its  greater  uniformity  it  is  being  more  and 
more  generally  used  for  routine  quantitative  work.  There  is  an  opti- 
mum reaction  for  every  variety  of  bacteria,  and  to  ensure  uniformity 
the  committee  of  the  American  Public  Health  Association  in  1897 
adopted  a  standard  which  was  as  near  as  possible  to  the  average  opti- 
mum for  water  bacteria.  Such  a  uniform  standard  is  a  necessity  to 
secure  comparability  of  the  results  of  various  observers.  At  best  only 
a  certain  proportion  of  bacteria  develop,  and  it  is  only  important  that 


BACTERIOLOGICAL  EXAMINATION  OF  WATER  445 

our  counts  represent  a  section  through  the  true  bacterial  flora  which 
fairly  represents  the  quick-growing  sewage  forms.  Comparability  is 
the  vitally  essential  factor. 

The  temperature  at  which  the  bacteria  develop  is  of  great  impor- 
tance, and  they  should  be  protected  from  light.  The  access  of  oxygen 
whicli  prevents  the  growth  of  anaerobes  must  also  not  be  forgotten. 
As  a  rule,  the  plate  cultures  are  developed  for  three  days  at  room  tem- 
perature, and  for  eighteen  hours  at  incubator  temperature.  Some 
bacteria  do  not  develop  colonies  in  three  days,  but  these  are  neglected. 
The  number  of  bacteria  growing  at  room  temperature  is  usually  five 
to  ten  times  as  many  as  at  35°  C. 

The  glucose  broth  is  placed  at  37°  C.  for  the  development  of  the  colon 
bacilli.  The  fermentation  tubes  not  showing  gas  are  recorded  and  usually 
discarded.  Those  showing  gas  are  suspected  to  contain  colon  bacilli. 
To  a  number  of  tubes  containing  melted  litmus-lactose  agar  at  about 
44°  C.  are  added  1,  0.1,  and  0.01  loop  of  the  culture  fluid.  Plates  are 
poured  and  the  whole  placed  in  the  incubator.  The  bacillus  coli  fer- 
ments lactose  and  thus  produces  acid ;  so  that  if  colon  bacilli  are  present 
we  have  a  number  of  red  colonies  on  a  blue  field.  Later,  if  many  colon 
bacilli  were  present,  the  whole  medium  becomes  acid.  At  forty-eight 
hours,  on  account  of  alkali  being  produced,  the  blue  color  returns. 

If  after  inspection  red  colonies  are  seen,  four  or  five  are  picked  and 
planted  into  glucose  bouillon  and  other  media.  For  the  characteristics 
of  the  colon  bacilli  the  Massachusetts  State  Board  of  Health  uses  six 
media — gelatin,  lactose  agar,  dextrose  broth,  milk,  nitrate  solution,  and 
peptone  solution. 

Significance  of  the  Colon  Bacillus. — The  colon  test  has  been  received 
by  the  majority  of  engineers  and  practical  sanitarians  with  great  satis- 
faction, and  has  been  applied  with  confidence  to  the  examination,  not 
only  of  water,  but  of  shell-fish  and  other  articles  of  food  as  well.  On 
the  other  hand,  some  have  denied  its  value.  Bacteriologists  have  found 
bacilli  like  certain  members  of  the  colon  group  in  apparently  unpol- 
luted well-water.  The  discovery  that  most  animals  have  colon  bacilli 
apparently  identical  in  the  usual  characteristics  studied  with  those  of 
man  has  complicated  matters.  Thus  a  fresh  hillside  stream  may  be 
loaded  with  colon  bacilli  from  the  washings  of  horse  or  cow  manure 
put  on  the  fields  through  which  it  runs,  or  polluted  by  a  stray  cow  or 
horse.  Swine,  hens,  birds,  etc.,  may  contaminate  in  unsuspected  WHYS. 
Up  to  the  present  there  is  no  conclusive  evidence  that  colon  bacilli 
increase  for  any  considerable  length  of  time  anywhere  except  in  the 
intestines  of  the  higher  vertebrates,  and  they  are  widely  distributed 
in  nature  mainly  because  the  fecal  discharges  of  man  and  animals  are 
a  common  thing  on  the  soil.  When  the  colon  bacillus  is  present  so  as 
to  be  isolated  from  1  c.c.  of  water  in  a  series  of  tests,  it  is  reasonable 
proof  of  pollution  and  the  conditions  should  be  investigated.  Ten 
colon  bacilli  in  1  c.c.  indicates  serious  pollution,  and  very  likely  a 
dangerous  one.  \Vinslow  reports  that  in  only  two  out  of  fifty-eight 
samples  of  presumably  non-polluted  waters  did  he  get  colon  bacilli  in 


446  BACTERIA  PATHOGENIC  TO  MAN 

the  1  c.c.  samples.  Even  in  twenty-one  stagnant  pools  he  only  found 
colon  bacilli  in  five  in  the  1  c.c.  samples. 

The  experience  of  all  who  have  practically  studied  the  subject  is 
that  in  delicacy  the  colon  test  surpasses  chemical  analysis;  in  constancy 
and  definiteness  it  also  excells  the  quantitative  bacterial  test. 

Interpretation  of  the  Quantitative  Analysis. — The  older  experimenters 
attempted  to  establish  arbitrary  standards  by  which  the  sanitary 
quality  of  water  could  be  fixed  automatically  by  the  number  of  germs 
alone.  This  has  been  largely  given  up.  Dr.  Sternberg  considers 
that  a  water  containing  less  than  100  bacteria  is  presumably  from  a 
deep  source  and  uncoritaminated  by  surface  drainage;  that  one  with  500 
bacteria  is  open  to  suspicion;  and  that  one  with  over  1000  bacteria  is 
presumably  contaminated  by  sewage  or  surface  drainage.  Even  this 
conservative  opinion  must  be  applied  with  caution.  The  source  of  the 
sample  is  of  vital  importance  in  the  interpretation;  thus,  a  bacterial 
count  which  would  condemn  a  spring  or  well  might  be  normal  for  a 
river.  In  woodland  springs  and  lakes  several  hundred  bacteria  are 
frequently  found.  In  lakes  the  point  at  which  the  sample  is  taken 
is  of  great  importance,  as  the  bacterial  count  varies  with  the  distance 
and  with  the  depth.  The  weather  also  is  an  influence,  since  the  wind 
causes  waves  which  stirs  up  the  bottom  mud.  Rains  greatly  influence 
streams  by  flooding  them  with  surface  water.  The  season  of  the  year 
is  an  important  factor.  The  counts  are  highest  in  the  winter  and  spring 
months,  and  lower  from  April  to  September. 

The  following  figures  illustrate  this  point: 

Water.                              Observer.  Year.  Jan.  Feb.  March.   April.    May.  June. 

New  York  City  tap-water.  Houghton  1904  890  1100  650      240      350  370 

Boston                      "  Whipple  1892  135  211  102        52        53  86 

Merrimac  River      "  Clark  1899  4900  5900  6300    2900     1900  3500 

The  spring  increase  is  not  an  exception  to  the  rule  that  high  num- 
bers indicate  danger  but  an  indication  of  its  truth,  for  it  means  a  melt- 
ing of  the  snow  and  a  flow  of  surface  water  into  the  streams.  A  number 
of  severe  epidemics  of  typhoid  fever  have  been  produced  in  this  way. 
Although,  as  a  rule,  a  series  of  tests  are  necessary  to  pass  judgment  on 
a  water,  a  single  test  may  be  very  important.  A  large  increase  in  the 
number  in  tap-water  a  day  after  a  storm  points  unerringly  to  surface 
pollution,  and  if  towns  exist  in  the  water-shed,  to  street  and  sewer 
pollution.  The  Croton  water  frequently  jumps  from  hundreds  to 
thousands  after  such  a  storm. 

In  a  typhoid  epidemic  at  Newport,  Winslow  reports  that  a  test  of 
the  water  supply  showed  but  334  bacteria  per  cubic  centimetre,  but 
one  from  a  well  showed  6100.  The  suspicion  aroused  was  justified  by 
finding  all  the  typhoid  cases  had  gotten  water  from  this  well. 

The  study  of  the  bacterial  effluent  from  municipal  water  filters  is 
the  only  way  in  which  the  efficiency  of  the  filter  and  the  accidents 
which  occur  can  be  determined.  In  Germany  these  regular  tests  are 
obligatory.  Elaborate  studies  have  recently  been  made  of  the  exact 


BACTERIOLOGICAL  EXAMINATION  OF  WATER  447 

distribution  of  streams  of  sewage  in  bodies  of  water  into  which  they 
flow,  their  disappearance  by  dilution  and  sedimentation,  and  their 
removal  by  death.  Under  peculiar  conditions  bacteria  in  water  may 
increase  for  a  time,  but  here  the  prevailing  bacteria  belong  almost 
exclusively  to  one  type. 

Streptococci  in  Sewage.— The  varieties  of  streptococci  found  most 
often  in  polluted  water  correspond  to  the  streptococci  described  by 
Houston.  In  some  water  in  which  these  are  found  no  B.  coli  have 
been  found  and  there  is  considerable  doubt  in  such  cases  as  to  whether 
the  streptococci  imply  serious  pollution.  The  streptococci  remain 
alive  much  longer  than  the  colon  bacilli,  and  therefore,  probably,  than 
the  typhoid  bacillus. 

The  Proteus  Group. — Members  of  the  proteus  group  are  often  found 
in  polluted  waters  and  but  rarely  in  pure  water.  The  bacillus  pyo- 
cyaneus  is  also  at  times  present,  and  has  in  a  few  cases  excited  infectious 
diarrhoea.  Bacillus  cloacae  is  a  common  form  of  sewage  bacteria. 

Isolation  of  the  Typhoid  Bacillus  from  Water. — If  it  were  possible 
to  readily  obtain  the  typhoid  bacilli  from  water,  when  they  were  present 
in  small  numbers,  its  examination  for  that  purpose  would  be  of  much 
greater  value  than  it  is  now;  but  we  have  to  remember  that  we  can 
only  examine  at  one  time  a  few  cubic  centimetres  of  water  by  bacterio- 
logical methods,  and  that  although  the  typhoid  bacilli  may  be  sufficiently 
abundant  in  the  water  to  give,  in  the  quantity  that  we  ordinarily  drink, 
a  few  bacilli,  yet  it  must  be  a  very  lucky  chance  if  they  happen  to  be 
in  the  small  amount  which  we  examine.  Still,  further,  although  it  is 
very  easy  to  isolate  typhoid  bacilli  from  water  when  they  are  in  con- 
siderable numbers,  yet  when  they  are  a  very  minute  proportion  of  all 
the  bacteria  present  it  is  almost  impossible  not  to  overlook  them.  Many 
attempts  have  been  made  to  devise  some  method  by  which  the  relative 
number  of  the  typhoid  and  other  parasitic  bacteria  present  in  water 
could  be  increased  at  the  expense  of  the  saprophytic  bacteria.  Thus 
to  100  c.c.  of  water  25  c.c.  of  a  4  per  cent,  peptone  nutrient  bouillon  is 
added,  and  the  whole  put  in  the  incubator  at  37°  C.  for  twenty-four 
hours.  From  this,  plate  cultures  are  made.  In  our  experience  this 
and  other  methods,  such  as  collecting  the  bacteria  left  on  a  filter  after 
passing  several  gallons  through,  have  not  enabled  us  to  detect  the 
typhoid  bacillus  where  we  have  failed  to  find  it  by  making  direct  plate 
cultures.  As  a  matter  of  fact,  the  typhoid  bacillus  is  rarely  found, 
even  in  specimens  of  water  where  we  actually  know  that  it  is  or  has 
been  present  because  of  cases  of  typhoid  fever  which  have  developed 
from  drinking  the  water.  From  these  facts  we  must  consider  our  lack 
of  finding  the  bacillus  in  any  given  case  as  absolutely  no  reason  for 
considering  the  water  to  be  free  from  danger.  Another  serious  draw- 
back to  the  value  of  the  examinations  for  the  typhoid  bacillus  is  that 
they  are  frequently  made  at  a  time  when  the  water  is  really  free  from 
contamination,  though  both  earlier  and  later  the  bacillus  was  present. 
It  is  hardly  worth  while,  therefore,  except  in  careful  experimental 
researches,  to  examine  the  water  for  the  typhoid  bacillus,  but  rather 


. 

448  BACTERIA  PATHOGENIC  TO  MAN 

study  the  location  of  the  surrounding  privies  and  sources  of  contamina- 
tion. A  number  of  observers,  resting  on  the  agglutination  test,  have 
thought  they  have  isolated  typhoid  bacilli  from  the  soil  and  water,  but 
these  investigators  had  not  considered  sufficiently  the  matter  of  group 
agglutinins,  and  their  results  are  not  trustworthy. 

Bacteriological  Examination  of  Air. 

Saprophytic  bacteria  are  always  present  in  considerable  numbers  in 
the  air  except  far  out  at  sea  or  on  high  mountains.  They  are  more 
abundant  where  organic  matter  abounds,  and  in  dry  and  windy  weather. 
Pathogenic  bacteria,  on  the  other  hand,  are  only  occasionally  present 
in  the  air.  The  practical  results  obtained  from  the  examination  of  air 
for  pathogenic  bacteria  have  been  slight.  We  know  that  at  times  they 
must  be  in  the  air,  but  unless  we  purposely  increase  their  numbers 
they  are  so  few  in  the  comparatively  small  amount  of  air  which  it  is 
practicable  to  examine  that  we  rarely  find  them.  Examination  of  dust, 
however,  in  hospital  wards  and  sick-rooms,  in  places  where  only  air 
infection  was  possible,  have  revealed  tubercle  bacilli  and  other  patho- 
genic bacteria. 

The  simplest  method  of  searching  for  the  varieties  of  bacteria  in  the 
air  and  their  number  in  any  place  is  to  expose  to  the  air  for  longer  or 
shorter  periods  nutrient  agar  spread  upon  the  surface  of  the  Petri  dish. 
After  exposure  the  plates  are  put  either  in  the  incubator  at  37°  C.  or 
kept  at  room  temperature.  The  more  careful  quantitative  examination 
is  made  by  drawing  a  given  quantity  of  air  through  tubes  containing 
sterile  sand,  which  is  kept  in  by  pieces  of  metal  gauze.  When  the 
operation  is  completed  the  sand  is  poured  into  a  tube  containing  melted 
nutrient  gelatin  or  nutrient  agar,  and  after  thoroughly  shaking,  the 
mixture  is  poured  into  a  Petri  dish  and  the  bacteria  allowed  to  develop, 
either  at  37°  or  20°  C.,  according  as  the  growth  of  the  parasitic  or  sapro- 
phytic  varieties  is  desired.  Instead  of  agar  or  gelatin  ascitic  broth  or 
animals  may  be  inoculated.  Such  examinations  are  occasionally  made 
of  the  air  of  theatres,  crowded  streets  in  cities,  etc.  They  give  inter- 
esting but  hardly  valuable  results. 

Bacteriological  Examination  of  the  Soil. 

The  subject  from  its  agricultural  side  cannot  be  considered  here. 
The  soil  can  be  gathered  in  sterile,  sharp-pointed,  sheet-iron  tubes.  As 
in  water,  we  wish  to  learn  the  number  of  bacteria  and  the  important 
varieties  of  bacteria  present.  To  estimate  the  number,  small  fractions 
of  a  gram  are  taken. 

According  to  Houston  uncultivated  sandy  soil  averages  100,000  bac- 
teria per  gram,  garden  soil  1,500,000,  and  sewage-polluted  115,000,000. 
The  most  important  bacteria  to  be  sought  for  are  bacilli  of  the  colon 
group  and  streptococci.  Both  of  these  suggest  fairly  recent  excremental 
pollution. 

The  period  during  which  typhoid  bacilli  remain  alive  in  soil  is  un- 


BACTERIOLOGICAL  EXAMINATION  OF  WATER  449 

known,  since  it  depends  on  so  many  unknown  factors  and  differs  so 
in  different  places.  The  typhoid  bacilli  probably  rarely  increase  in 
the  soil  and  probably  rarely  survive  a  month  in  it  The  main  danger 
of  soil  bacteria  is  their  being  washed  in  water  supplies  by  rains  and 
wind. 

Contamination  and  Purification  of  Drinking  Waters. 

Brook-water  and  river-water  are  contaminated  in  two  ways:  through 
chemicals,  the  waste  products  of  manufacturing  establishments,  and 
through  harmful  bacteria  by  the  contents  of  drains,  sewers,  etc.,  the 
latter  method  being  by  far  the  more  dangerous. 

When  water,  which  has  been  soiled  by  waste  products  of  manufac- 
tories only,  becomes  so  diluted  or  purified  that  the  contamination  is 
not  noticeable  to  the  senses  and  shows  no  dangerous  products  on  chemi- 
cal analysis,  it  is  probably  safe  to  drink.  When  sewage  is  the  contami- 
nation this  rule  no  longer  holds,  and  there  may  be  no  chemical  impuri- 
ties and  no  pathogenic  bacteria  found,  and  yet  disease  be  produced. 
That  river-water  which  has  been  fouled  by  sewage  will,  in  the  course 
of  a  few  miles,  through  the  dilution  of  additional  supplies,  through 
sedimentation,  and  through  oxidation,  become  greatly  purified  is  an 
indisputable  fact.  The  increase  in  bacteria  which  occurs  from  con- 
tamination is  also  largely  or  entirely  lost  after  ten  to  twenty  miles  of 
river  flow.  Nevertheless,  the  history  of  many  epidemics  seems  to  show 
that  a  badly  contaminated  river  is  never  an  absolutely  safe  water  to 
drink,  although  with  the  lapse  of  each  day  it  becomes  less  and  less 
dangerous,  nor  will  sand  filter  beds  absolutely  remove  all  danger. 
These  statements  are  founded  upon  the  results  of  numerous  investiga- 
tions; thus  the  marked  disappearance  of  bacteria  is  illustrated  by  the 
following:  Kummel  found  below  the  town  of  Rosbock  48,000  bacteria 
to  the  cubic  centimetre;  twenty-five  kilometres  farther  down  the  stream 
only  200  were  present — about  the  same  number  as  before  the  sewage 
of  Rosbock  entered.  On  the  other  hand,  the  doubtful  security  of 
depending  on  a  river  purification  is  proved  by  such  experiences  as  the 
following:  In  the  city  of  Lowell,  Massachusetts,  an  alarming  epidemic 
followed  the  pollution  of  the  Merrimac  River  three  miles  above  by 
typhoid  feces,  and  six  weeks  later  an  alarming  epidemic  attacked  Law- 
rence, nine  miles  below  Lowell.  It  was  estimated  that  the  water  took 
ten  days  to  pass  from  Lowell  to  Lawrence  and  through  the  reservoirs. 
Typhoid  bacilli  usually  die  in  river-water  in  from  three  to  ten  days, 
but  they  may  live  for  twenty-five  days  in  other  water;  the  Lawrence 
epidemic  is  easily  explained.  Newark-on-Trent,  England,  averaged 
seventy-five  cases  *a  year  from  filtered  water  and  only  ten  when  it  was 
changed  to  deep-well  supply. 

Purification  of  Water  on  a  Large  Scale. — For  detailed  information 
on  this  subject  the  reader  is  referred  to  works  on  hygiene.  Surface 
waters,  if  collected  and  held  in  sufficiently  large  lakes  or  reservoirs 
usually  beome  so  clarified  by  sedimentation,  except  shortly  after  heavy 
rains,  *as  to  require  no  further  treatment  so  far  as  its  appearance  goes. 

29 


450  BACTERIA  PATHOGENIC  TO  MAN 

The  collection  of  water  in  large  reservoirs  allows  not  only  the  living 
and  dead  matter  to  subside,  but  allows  time  also  for  the  pathogenic 
germs  to  perish  through  light  and  antagonistic  bacteria  and  other 
deleterious  influences,  sand  or  mechanical  coagulant.  Filtration  of 
water  exerts  a  very  marked  purification,  taking  out  99.8  per  cent,  of  the 
organisms  in  those  best  constructed  and  at  least  95  per  cent,  in  those 
cpmmonly  used  in  cities.  The  construction  of  filters  is  too  large  a  sub- 
ject to  enter  on  minutely  here;  sand  filters  consist,  as  a  rule,  of  several 
layers,  beginning  with  fine  sand,  and  then  smaller  and  larger  gravel, 
and  finally  rough  stones.  A  certain  time  elapses  before  the  best  results 
are  obtained;  this  seems  to  wait  for  the  formation  of  a  film  of  organic 
material  on  the  sand,  which  is  full  of  nitrifying  bacteria.  Even  the 
best  filters  only  greatly  diminish  the  dangers  of  polluted  water.  Spring- 
water  and  well-water  are,  in  fact,  filtered  waters. 

Water  which  is  subject  to  serious  pollution  must  be  submitted  to  a 
preliminary  purification  before  it  can  be  considered  a  suitable  source 
for  a  drinking-water  supply.  The  means  employed  for  its  purification 
depend  to  a  large  extent  upon  the  character  of  the  water  and  the  nature 
of  the  pollution.  Filtration  through  slowT  sand  filters,  three  to  five 
feet  in  depth,  removes  98  to  99.5  per  cent,  of  the  bacteria  and  organic 
matter;  so  that  effluents  from  the  best  constructed  sand  filtration  beds 
constitute  safe  and  reliable  drinking  waters.  Five  hundred  thousand 
to  one  or  two  million  gallons,  depending  somewhat  upon  the  extent  of 
pollution  and  the  fineness  of  the  sand,  can  be  filtered  daily  per  acre. 
Only  the  surface  of  the  sand  filter  becomes  in  any  way  clogged  and 
as  thin  a  layer  as  can  be  scraped  off  is  removed  one  or  more  times  a 
month.  This  surface  sand  is  washed  with  clean  water  and  several 
scrapings  replaced  at  one  time.  Sand  filtration  beds  are  very  widely 
used  abroad  and  are  coming  into  extensive  use  in  this  country.  The 
filter  beds  at  Lawrence,  Mass.,  have  been  used  over  ten  years  with 
marked  success,  rendering  the  highly  polluted  Merrimac  River  a  safe 
drinking-water;  the  filter  beds  there  are  scraped  about  thirteen  times 
a  year. 

Mechanical  filtration  plants  find  considerable  favor  where  clarifica- 
tion as  well  as  bacterial  purification  is  desired.  A  coagulant  such  as 
sulphate  of  aluminum  is  employed  and  forms  in  the  water  a  flocculent 
precipitate  which  carries  down  with  it  all  suspended  matter;  50,000,000 
or  more  gallons  of  water  can  be  filtered  on  an  acre  daily,  but  the  filters 
must  be  washed  once  or  twice  daily  by  reversing  the  flow  and  cleansing 
the  clogged  filter  with  a  stream  of  the  purified  water.  The  bad  clogging 
is  due  to  the  fact  that  the  process  is  a  purely  mechanical  one,  and  not 
comparable  in  any  way  with  the  vital  processes  carried  on  in  the  sand 
filter  by  the  nitrifying  bacteria. 

Other  methods  are  coming  into  use,  such  as  the  passage  of  ozone, 
and  have  proved  successful.  Such  processes  should  be  under  the  direct 
supervision  of  expert  sanitary  engineers  and  bacteriologists. 

Domestic  Purification. — Water  which  requires  private  filtering  should 
not  be  supplied  for  drinking  purposes.  Unhappily,  however,  it  often 


BACTERIOLOGICAL  EXAMINATION  OF  WATER  451 

is.  Filters  may  be  divided,  roughly,  into  those  for  low  and  high  pressure. 
The  former  are  directly  connected  with  the  water  main,  while  the 
others  simply  have  the  slight  pressure  of  the  column  of  water  stand- 
ing in  the  filter.  Many  high-pressure  filters  contain  animal  cha renal, 
silicate*!  carbon,  etc.,  either  in  a  pressed  condition  or  in  one  porous  in 
These  filters  remove  much  of  the  deleterious  matter  from  the  suspected 
waters,  but  the  majority  cannot  be  depended  upon  to  remove  all  bacteria. 
Even  those  which  are  equipped  for  self-cleansing  become  in  a  little  while 
foul,  and,  if  not  cleaned,  unfit  for  use.  The  best  of  the  class  are  of  porous 
stone,  such  as  the  Berkefeld  and  Pasteur  filters.  These  yield  a  water,  if 
too  great  pressure  is  not  used,  almost  absolutely  free  from  bacteria,  and  if 
they  are  frequently  cleansed  they  are  reliable.  A  large  Berkefeld  filter 
will  allow  sixty  gallons  of  water  to  pass  per  hour.  The  Pasteur  filter 
is  more  compact  and  slower.  From  the  best  Pasteur  filters  sterile  water 
may  be  passed  for  two  to  three  weeks;  from  the  Berkefeld  usually  only 
a  few  days.  A  simple  typical  low-pressure  filter  is  that  of  Bailey  Den- 
ton.  The  upper  compartment  contains  the  filtering  material,  which 
may  be  sand  or  charcoal,  and  is  fed  from  a  cistern  or  hydrant.  After 
a  certain  cjuantity  of  water  has  passed  in,  the  supply  is  automatically 
cut  off  until  the  whole  amount  has  filtered.  A  filter  easily  made  is  the 
following:  Take  a  large-sized  earthenware  pot  and  plug  the  hole  in 
the  bottom  with  a  cork,  through  which  pass  a  short  glass  tube.  Upon 
the  bottom  place  an  inch  of  small  pieces  of  broken  flower-pot;  upon 
this  a  couple  of  inches  of  well-washed  small  gravel,  and  upon  this  six 
to  twelve  inches  of  well-washed,  fine,  sharp  sand.  Cover  the  sand  with 
a  piece  of  filter  paper  and  hold  this  down  with  a  few  small  stones. 
Mount  the  pot  on  a  tripod,  and  it  is  ready  for  use.  The  paper  prevents 
the  sand  being  disturbed  when  water  is  added,  and  as  it  also  holds 
most  of  the  sediment,  this  can  be  readily  removed.  Every  few  months 
the  sand  can  be  washed  and  replaced.  Animal  charcoal  is  not  a  good 
substance  for  permanent  filters,  as  bacteria  grow  well  in  it.  Whenever 
water  is  suspected,  and  there  is  any  doubt  as  to  the  filters,  it  should^be 
boiled  for  ten  minutes;  this  will  destroy  all  bacteria.  This  precaution 
should  always  be  taken  in  the  presence  of  typhoid  fever  and  cholera 
epidemics. 

The  Disposal  of  Sewage. 

The  disposal  of  sewage  is  becoming  a  vital  question  with  all  towns 
and  cities  which  are  not  situated  near  salt-water  outlets,  since  the 
present  tendency  in  legislation  is  to  compel  such  towns  to  dispose  of 
their  waste  so  that  it  shall  not  be  a  menace  to  drinking-water  streams, 
destructive  to  fisheries,  or  a  nuisance  to  harbors. 

Methods  of  sewage  purification  depends  upon  the  character  of  the 
sewage  and  the  kind  of  effluent  desired. 

Two  hundred  thousand  gallons  of  crude  sewage  may  be  filtered 
upon  an  acre  of  land  daily  and  an  effluent  obtained  which  will  com- 
pare favorably  in  every  way  known  to  the  chemist  and  bacteriologists 
with  the  best  "mountain  springs.  This  is,  however,  a  slow  process  and 


452  BACTERIA  PATHOGENIC  TO  MAN 

it  is  rare  that  such  a  pure  effluent  is  required.  Similar  results  may  be 
obtained  by  utilizing  the  septic  tank  method,  running  the  sewage  from 
the  septic  tank  to  contact  beds  and  thence  to  sand  filter  beds;  where 
because  of  the  partial  "self-purification  of  the  sewage"  in  the  septic  tank 
and  contact  beds  2,500,000  gallons  of  sewage  can  be  filtered  daily  on 
an  acre  of  surface.  In  this  process  less  land  is  required  and  both  these 
effluents  can  be  safely  turned  into  drinking-water  streams. 

If,  however,  a  merely  non-putrescible  effluent  is  required,  one  which, 
though  high  bacterially,  will  not  be  offensive  in  any  way,  or  subject  to 
further  decomposition,  it  may  be  obtained  by  passing  crude  sewage 
to  septic  tanks,  thence  to  double  contact  beds,  the  resulting  effluent 
having  merely  an  earthy,  humus-like  odor  and  being  non-putrescible. 

Where  acid  wastes,  tannery  wastes,  dyestuffs,  etc.,  from  various 
factories  enter  into  sewage,  its  disposal  becomes  a  more  complicated 
problem  and  chemical  precipitation  by  the  use  of  lime  or  other  chemi- 
cals is  generally  employed  for  such  sewage  purification,  which  at  best 
is  only  partial  and  is  sometimes  supplemented  by  sand  filtration. 


CHAPTER   XXXVI. 

THE  BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE. 

FROM  the  standpoint  of  the  dairy  and  cheese  producer  a  study  of  the 
different  varieties  of  bacteria  of  the  air  and  dust  are  of  importance. 
Some  yield  products  which  give  the  butter,  cheese,  milk,  or  cream 
a  bad  taste  or  odor.  We  can  only  consider  here  the  bacteriology  of 
milk  so  far  as  it  is  related  to  health  and  disease. 

The  bacteria  in  milk  can  be  divided  into  two  great  groups — those 
which  get  into  the  milk  after  it  leaves  the  udder  and  those  which 
come  from  the  cow.  The  first  group  comprises  bacteria  from  dust,  etc. 

The  extraneous  bacteria  are  of  importance  because  they  produce 
changes  in  the  chemical  composition  of  the  milk  when  they  have  devel- 
oped in  great  numbers.  The  number  of  bacteria  in  any  sample  of 
milk  depends  on  three  factors :  the  number  deposited  in  the  milk  from 
the  cow's  udder,  from  the  air,  and  utensils ;  the  time  during  which  they 
have  developed,  and  the  temperature  at  which  the  milk  has  stood. 
The  last  is  perhaps  the  most  important.  The  attempt  was  made  dur- 
ing the  past  three  years  to  connect  illness  in  children  with  the  varieties 
of  bacteria  in  milk.  As  a  matter  of  fact  no  such  connection  was  made 
out.  The  number  of  varieties  was  found  to  be  so  great  that  if  any 
special  type  of  disease  had  developed  in  those  getting  milk  it  would 
have  been  an  extremely  difficult  thing  to  have  made  out  such  a  connec- 
tion.1 This  in  spite  of  the  fact  that  the  varieties  isolated  represent  only 
the  species  present  in  greatest  number  in  the  milk  examined,  for  in  no 
case  was  more  than  0.01  c.c.  of  a  milk,  and  in  most  highly  contaminated 
milks  only  0.001  c.c.,  used  in  making  a  plate,  and  varieties  which 
occurred  in  too  small  numbers  to  be  present  in  this  quantity  would 
necessarily  be  missed.  For  the  purposes  of  this  book  it  is  not  con- 
sidered desirable  to  burden  the  reader  with  the  enumeration  of  the 
varieties  of  bacteria  found  in  the  different  samples  of  milk  and  their 
characteristics.  Only  a  brief  summary  of  the  results  will  be  given. 

1  The  bacteria  were  isolated  from  the  milk  through  plating  in  a  2  per  cent,  lactose-litmus  nutrient 
gelatin  or  agar,  and  later  grown  upon  the  usual  identification  media.  The  pathogenic  properties  of 
the  different  bacteria  were  tested  by  intraperitoneal  and  subcutaneous  inoculation  in  guinea-pigs 
with  2  c.c.  of  a  forty-eight-hour  broth  culture,  and  by  feeding  young  kittens  for  several  days  with 
3  to  6  c.c.  daily  of  a  twenty-four-hour  broth  culture  by  means  of  a  medicine  dropper. 

With  the  characteristics  of  the  bacteria  thus  determined,  they  were  then  separated  into  classes 
following  as  nearly  as  possible  the  lines  suggested  in  Chester's  Manual  of  Determinative  Bacteri- 
ology. Further  attempt  was  then  made  to  identify  as  many  as  possible  of  the  varieties  with  those 
previously  described,  using  the  descriptions  of  Chester  and  Migula.  With  a  great  many  this  proved 
unsatisfactory  or  impossible  because  cf  the  incomplete  descriptions  in  literature,  or  the  lack  of  all 
description. 


454  BACTERIA  PATHOGENIC  TO  MAN 

From  the  milks  altogether,  239  varieties  of  bacteria  were  isolated 
and  studied.  These  239  varieties,  having  some  cultural  or  other  differ- 
ences, were  divided  into  the  31  classes,  each  class  containing  from  1 
to  39  more  or  less  closely  related  organisms. 

As  to  the  sources  of  bacteria  found  in  milk,  we  made  sufficient  experi- 
ments to  satisfy  us  that  they  came  chiefly  from  outside  the  udder  and 
milk-ducts. 

Bacteria  were  isolated  from  various  materials  which  under  certain 
conditions  might  be  sources  of  contamination  for  the  milk,  and  the 
cultures  compared  with  those  taken  from  milk.  Thus  there  were 
obtained  from  20  specimens  of  hay  and  grass,  31  varieties  of  bacteria; 
from  15  specimens  of  feces,  manure,  and  intestinal  contents,  28  varieties; 
from  10  specimens  of  feed,  17  varieties.  Of  these  76  varieties  there 
were  26  which  resembled  closely  those  from  milk — viz.,  11  from  grass 
or  hay;  26  from  manure;  5  from  feed. 

During  the  investigation  a  number  of  the  varieties  isolated  from 
milk  were  shown  to  be  identical  with  types  commonly  found  in  water. 

From  the  few  facts  quoted  above  and  from  many  other  observations 
made  during  the  course  of  the  work,  it  would  seem  that  the  term  "milk 
bacteria"  assumes  a  condition  which  does  not  exist  in  fact.  The  expres- 
sion would  seem  to  indicate  that  a  few  varieties,  especially  those  derived 
in  some  way  from  the  cow,  are  commonly  found  in  milk,  which  forms 
having  entered  the  milk  while  still  in  the  udder,  or  after  its  withdrawal, 
are  so  well  fitted  to  develop  in  milk  that  they  overgrow  all  other  varieties. 

As  a  matter  of  fact,  it  was  found  that  milk  taken  from  a  number  of 
cows,  in  which  almost  no  outside  contamination  had  occurred,  and 
plated  immediately,  contained,  as  a  rule,  very  few  bacteria,  and  these 
were  streptococci,  staphylococci,  and  other  varieties  of  bacteria  not 
often  found  in  milk  sold  in  New  York  City;  the  temperature  at  which 
milk  is  kept  being  less  suitable  for  them  than  for  the  bacteria  which  fall 
into  the  milk  from  dust,  manure,  etc.  A  number  of  specimens  of  fairly 
fresh  market  milk  averaging  200,000  bacteria  per  cubic  centimetre  were 
examined  immediately,  and  again  after  twelve  to  twenty-four  hours.  In 
almost  every  test  the  three  or  four  predominant  varieties  of  the  fresher 
milk  remained  as  the  predominant  varieties  after  the  period  mentioned. 

The  above  experiments  seem  to  show  that  organisms  which  have 
gained  a  good  percentage  in  the  ordinary  commercial  milk  at  time  of 
sale  will  be  likely  to  hold  the  same  relative  place  for  as  long  a  period 
as  milk  is  usually  kept.  After  the  bacteria  pass  the  ten  or  twenty 
million  mark  a  change  occurs,  since  the  increasing  acidity  inhibits  the 
growth  of  some  forms  before  it  does  that  of  others.  Thus  some  varieties 
of  the  lactic  acid  bacteria  can  increase  until  the  acidity  is  twice  as  great 
as  that  which  inhibits  the  growth  of  streptococci.  Before  milk  reaches 
the  curdling  point,  the  bacteria  have  usually  reached  over  a  billion  to 
each  cubic  centimetre.  For  the  most  part  specimens  of  milk  from  different 
localities  showed  a  difference  in  the  character  of  the  bacteria  present,  in 
the  same  way  that  the  bacteria  from  hay,  feed,  etc.,  varied.  Even  the 
intestinal  contents  of  cows,  the  bacteriology  of  which  might  be  expected 


BACTERIOLOGY  OF  MILK  IX  ITS  RELATION  TO  DISEASE     -4:,:, 

to  show  common  characteristics,  contained,  besides  the  predominating 
colon  types,  other  organisms  which  differed  widely  in  different  spe< -i« •- 
and  in  different  localities.  Cleanliness  in  handling  the  milk  and  the 
temperature  at  which  it  had  been  kept  were  also  found  to  have  a 
marked  influence  on  the  predominant  varieties  of  bacteria  present. 

Pathogenic  Properties  of  the  Bacteria  Isolated. — Intraperitoneal  injec- 
tion of  2  c.c.  of  broth  or  milk  cultures  of  about  40  per  cent,  of  the  varieties 
tested  caused  death.  Cultures  of  most  of  the  remainder  produced  no 
apparent  deleterious  effects  even  when  injected  in  larger  amounts. 
The  filtrates  of  broth  cultures  of  a  number  of  varieties  were  tested, 
but  only  one  was  obtained  in  which  poisonous  products  were  abun- 
dantly present.  Death  in  guinea-pigs  weighing  300  grams  followed 
within  fifteen  minutes  after  an  injection  of  2  c.c.;  1  c.c.  had  little  effect. 

As  bacteria  in  milk  are  swallowed  and  not  injected  under  the  skin, 
it  seemed  wise  to  test  the  effect  of  feeding  them  to  every  young  animals. 
We  therefore  fed  forty-eight-hour  cultures  of  139  varieties  of  bacteria 
to  kittens  of  two  to  ten  days  of  age  by  means  of  a  glass  tube.  The 
kittens  received  5  to  10  c.c.  daily  for  from  three  to  seven  days.  Only 
one  culture  produced  illness  or  death.  A  full  report  on  the  identifica- 
tion of  the  varieties  of  bacteria  met  with  in  this  investigation  can  be 
found  in  an  article  by  Dr.  Letchworth  Smith  in  the  1902  Annual 
Report  of  the  Department  of  Health. 

After  three  years  of  effort  to  discover  some  relation  between  special 
varieties  of  bacteria  found  in  milk  and  the  health  of  children  the  con- 
clusion has  been  reached  that  neither  through  animal  tests  nor  the 
isolation  from  the  milk  of  sick  infants  have  we  been  able  to  establish 
such  a  relation.  Pasteurized  or  "sterilized"  milk  is  rarely  kept  in 
New  York  longer  than  thirty-six  hours,  so  that  varieties  of  bacteria 
which  after  long  standing  develop  in  such  milk  did  not  enter  into  our 
problem.  The  harmlessness  of  cultures  given  to  healthy  young  kittens 
does  not  of  course  prove  that  they  would  be  equally  harmless  in  infants. 
Even  if  harmless  in  robust  infants,  they  might  be  injurious  when  sum- 
mer heat  and  previous  disease  had  lowered  the  resistance  and  the 
digestive  power  of  the  subjects.  In  a  recent  investigation  by  Dr.  D.  H. 
Bergey  some  connection  between  diarrhoea  and  pus  and  streptococci 
were  found. 

The  results  of  this  investigation  appear  to  warrant  the  following 
conclusions : 

1 .  The  occurrence  of  pus  in  cows'  milk  is  probably  always  associated 
with  the  presence  in  the  udder  of  some  inflammatory  reaction  brought 
about  by  the  presence  of  some  of  the  ordinary  pyogenic  bacteria,  espe- 
cially of  streptococci. 

2.  When  a  cow's  udder  has  once  become  infected  with  the  pvnirenic 
bacteria  the  disease  tends  to  persist  for  a  long  time,  probably  extending 
over  several  periods  of  lactation. 

3.  Lactation  has  no  causative  influence  per  se  upon  the  cellular  and 
bacterial  content  of  cows'  milk,  though  it  probably  tends  toward  the 
aggravation  of  the  disease  when  the  udder  is  once  infected. 


456  BACTERIA  PATHOGENIC  TO  MAN 

4.  The  so-called  "gelbe  gait,"  or  contagious  mammitis  of  European 
writers,  appears  to  be  merely  a  severe  form  of  mammitis  due  to  a  variety 
of  streptococcus  which,  on  account  of  its  chromogenic  properties,  gives 
to  the  milk  its  peculiar  golden-yellow  color. 

Our  failure  to  discover  definite  pathogenic  bacteria  in  milk,  as  well 
as  the  numerous  varieties  of  bacteria  met  with,  have  forced  us  to  rely 
on  the  clinical  observation  of  infants  to  note  what  difference,  if  any, 
occurred  in  those  fed  on  raw  and  Pasteurized  milk  from  the  same  source, 
and  upon  different  milks  of  unknown  origin  varying  in  the  number  of 
bacteria  contained.  These  observations  were  combined  with  those  upon 
other  factors  which  influenced  the  health  of  the  infants. 

Heated  Milk  vs.  Raw  Milk  for  Infants. — During  each  of  the  summers 
of  1902,  1903,  and  1904  a  special  lot  of  milk  was  modified  at  one  of  the 
Straus  depots  for  a  group  of  fifty  infants,  all  of  whom  were  under  nine 
months  of  age,  and  distributed  daily  in  the  usual  way.  To  one-half  the 
infants  the  milk  was  given  raw;  to  the  other  half  Pasteurized. 

The  modified  milk  was  made  from  a  fairly  pure  milk  mixed  with 
ordinary  cream.  The  bacteria  contained  in  the  milk  numbered  on 
the  average  45,000  per  cubic  centimetre,  in  the  cream  30,000,000.  The 
modified  raw  milk  taken  from  the  bottles  in  the  morning  averaged 
1,200,000  bacteria  per  cubic  centimetre,  or  considerably  less  than  the 
ordinary  grocery  milk;  the  Pasteurized,  about  1000;  taken  in  the  late 
afternoon  of  the  same  day  they  had  respectively  about  20,000,000  and 
50,000. 

Twenty-one  predominant  varieties  of  bacteria  were  isolated  from 
six  specimens  of  this  milk  collected  on  different  days.  The  varieties 
represented  the  types  of  bacteria  frequently  found  in  milk.  The  infants 
were  selected  during  the  first  week  in  June,  and  at  first  all  were  placed 
on  Pasteurized  milk.  The  fifty  infants  which  had  been  selected  were 
now  separated  into  two  groups  as  nearly  alike  as  possible.  On  the  15th 
of  June  the  milk  was  distributed  without  heating  to  one-half  the  infants, 
the  other  half  receiving  as  before  the  heated  milk.  In  this  way  the 
infants  in  the  two  groups  received  milk  of  identically  the  same  quality, 
except  for  the  changes  produced  by  heating  to  165°  F.  for  thirty  minutes. 
The  infants  were  observed  carefully  for  three  months  and  medical 
advice  was  given  when  necessary.  When  severe  diarrhoea  occurred 
barley-water  was  substituted  for  milk. 

The  first  season's  trial  gave  the  following  results:  Within  one  week 
20  out  of  the  27  infants  put  on  the  raw  milk  suffered  from  moderate 
or  severe  diarrhoea;  while  during  the  same  time  only  5  cases  of  moderate 
and  none  of  severe  diarrhoea  occurred  in  those  taking  Pasteurized  milk. 
Within  a  month  8  of  the  27  had  to  be  changed  from  raw  back  to  heated 
milk,  because  of  their  continued  illness;  7,  or  25  per  cent.,  did  well  all 
summer  on  raw  milk.  On  the  other  hand,  of  those  receiving  the  Pas- 
teurized milk,  75  per  cent,  remained  well,  or  nearly  so,  all  summer, 
while  25  per  cent,  had  one  or  more  attacks  of  severe  diarrhoea.  There 
were  no  deaths  in  either  group  of  cases. 

During  the  second  summer  a  similar  test  was  made' with  45  infants. 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     457 

Twenty-four  were  put  on  raw  modified  milk;  13  of  these  had  serious 
diarrhoea,  in  5  of  whom  it  was  so  severe  that  they  were  put  back  upon 
heated  milk;  10  took  raw  milk  all  summer  without  bad  effects;  2  died, 
1  from  gross  neglect  on  the  part  of  the  mother,  the  other  from  diarrhoea. 
Of  the  21  on  Pasteurized  milk,  5  had  severe  attacks  of  diarrhoea,  but 
all  were  kept  on  this  milk  except  for  short  periods,  when  all  food  was 
omitted;  16  did  well  throughout  the  summer.  One  infant,  markedly 
rachitic,  died.  The  third  summer's  results  have  not  been  tabulated, 
but  were  similar  to  those  of  the  first  two  tests. 

The  outcome  of  these  observations  during  the  first  two  summers  are 
summarized  in  the  following  table: 


Kind  of  milk. 

Num- 
ber of 
infants 

Re- 
mained 
well  for 
entire 
sum- 
mer. 

Number 
having 
severe  or 
moderate 
diarrhoea. 

Average 
number 
days  off 
milk  dur- 
ing sum- 
mer. 

Average 
weekly- 
gain  in 
weight. 

Aver'ge 
number 
of  days 
diar- 
rhoea. 

Deaths. 

Pasteurized  milk,  1000  to  50,000  ) 
bacteria  per  c.c.  j 

41 

31 

10 

3 

4.  0  oz. 

3.9 

1 

Raw  milk,  1.200,000  to  20,000,000  ) 
bacteria  per  c.c.  j 

5H 

17 

33 

5.5 

3.5    " 

11.5 

2 

Although  the  number  of  cases  was  not  large,  the  results,  almost 
identical  during  the  three  summers,  indicate  that  even  a  fairly  pure  milk 
when  given  raw,  in  hot  weather,  causes  illness  in  a  much  larger  per- 
centage of  cases  than  the  same  milk  given  after  Pasteurization.  A 
considerable  percentage  of  infants,  however,  do  apparently  quite  as 
well  on  raw  as  on  Pasteurized  wilk. 

Heated  Milk  vs.  Raw  Milk  for  Older  Children. — The  children  over  three 
years  of  age  who  received  unheated  milk,  containing  at  different  times 
from  145,000  to  350,000,000  bacteria  per  cubic  centimetre,  showed 
almost  no  gastrointestinal  disturbance.  The  conditions  at  three  insti- 
tutions will  serve  as  examples. 

In  the  first  of  these  an  average  grade  of  raw  milk  was  used  which, 
during  the  summer  contained  from  2,000,000  to  30,000,000  bacteria 
per  cubic  centimetre.  This  milk  was  stored  in  an  ice-box  until  re- 
quired. It  was  taken  by  the  children  unheated  and  yet  no  case  of  diar- 
rhoea of  sufficient  gravity  to  send  for  a  physician  occurred  during  the 
entire  summer.  This  institution  was  an  orphan  asylum  containing  650 
children  from  three  to  fourteen  years  of  age — viz.,  three  to  five  years,  98; 
five  to  eight  years,  162;  eight  to  fourteen  years,  390. 

A  second  institution  used  an  unheated  but  very  pure  milk  which  was 
obtained  from  its  own  farm.  This  milk  averaged  50,000  bacteria  per 
cubic  centimetre.  The  inmates  were  70  children  of  ages  ranging  from 
three  to  fourteen  years.  In  this  institution  not  a  single  case  of  diar- 
rhoeal  disease  of  any  importance  occurred  during  the  summer. 

1  Thirteen  of  the  fifty-one  infants  on  raw  milk  were  transferred  before  the  end  of  the  trial  to 
Pasteurized  milk  because  of  serious  illness.  If  these  infants  had  been  left  on  raw  milk  it  is  believed 
by  the  writers  that  the  comparative  results  would  have  been  even  more  unfavorable  to  raw  milk. 


458  BACTERIA  PATHOGENIC  TO  MAN 

In  a  third  institution  an  average  grade  of  milk  was  used  which  was 
heated.  This  milk  before  heating  contained  2,000,000  to  20,000,000 
bacteria  per  cubic  centimetre.  The  institution  was  an  infant  asylum 
in  which  there  were  126  children  between  the  ages  of  two  and  five  years. 
There  were  no  cases  of  diarrhoea  during  the  summer. 

These  clinical  observations  taken  in  connection  with  the  bacterio- 
logical examination  at  the  laboratory  show  that  although  the  milk 
may  come  from  healthy  cattle  and  clean  farms,  and  be  kept  at  a  tem- 
perature not  exceeding  60°  F.,  a  very  great  increase  in  the  number  of 
bacteria  may  occur.  Furthermore,  this  may  occur  without  the  accu- 
mulation in  'the  milk  of  sufficient  poisonous  products  or  living  bacteria 
to  cause  appreciable  injury  in  children  over  three  years  of  age,  even 
when  such  milk  is  consumed  in  considerable  amount  and  for  a  period 
extending  over  several  months.  Milk  kept  at  temperatures  somewhat 
above  60°  F.  was  not  met  with  in  our  investigations,  but  the  histories 
of  epidemics  of  ptomain  poisoning  teach  that  such  milk  may  be  very 
poisonous.  It  is  also  to  be  remembered  that  milk  abounding  in  bac- 
teria on  account  of  its  being  carelessly  handled  is  also  always  liable  to 
contain  pathogenic  organisms  derived  from  human  or  animal  sources. 

Results  with  Very  Impure  Milk  Heated  vs.  Those  with  Pure  or  Average 
Milk  Heated. — During  the  summer  of  1901  we  were  able  to  observe  a 
number  of  babies  fed  on  milk  grossly  contaminated  by  bacteria.  In 
1902,  systematic  supervision  of  all  stores  selling  milk  was  instituted  by 
the  Health  Department,  so  that  the  very  worst  milk  was  not  offered 
for  sale  that  summer. 

The  observations  upon  the  impure  milk  of  1901  are  of  sufficient 
importance  to  be  given  in  detail,  although  already  mentioned  in  the 
report  of  the  observations  upon  infants  of  both  summers  which  were 
fed  on  "store  milk."  A  group  of  over  150  infants  was  so  divided  that 
20  per  cent,  were  allowed  to  remain  on  the  cheapest  store  milk  which 
they  were  taking  at  the  time.  To  about  the  same  number  was  given 
a  pure  bottled  milk.  A  third  group  was  fed  on  the  same  quality  of 
milk  as  the  second,  but  sterilized  and  modified  at  the  Good  Samaritan 
Dispensary.  A  fourth  group  received  milk  from  an  ordinary  dairy  farm. 
This  milk  was  sent  to  a  store  in  cans  arid  called  for  by  the  people.  A 
few  infants  fed  on  breast  and  condensed  milk  were  observed  for  control. 

In  estimating  the  significance  of  the  observations  recorded  in  the 
tables,  one  should  bear  in  mind  that  not  only  do  different  infants  possess 
different  degrees  of  resistance  to  disease,  but  that,  try  as  hard  as  the 
physicians  could,  it  was  impossible  to  divide  the  infants  into  groups 
which  secured  equal  care,  and  were  subjected  to  exactly  the  same  con- 
ditions. It  was  necessary  to  have  the  different  groups  in  somewhat 
different  parts  of  the  city.  It  thus  happened  that  the  infants  on  the 
cheap  store  milk  received  less  home  care  than  the  average,  and  that 
those  on  the  pure  bottled  milk  lived  in  the  coolest  portion  of  the  city. 
Certain  results  were,  however,  so  striking  that  their  interpretation  is 
fairly  clear.  It  is  to  be  noted  that  the  number  of  infants  included  in 
each  group  is  small. 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     459 


TABLE  SHOWING  THE  RESULTS  OF  FEEDING  DURING  JULY  AND  AUGUST,  1901, 
IN  TENEMENT  HOUSES,  OF  112  BOTTLE-FED  INFANTS  UNDER  1  YEAR  OF  AGE, 
AND  OF  47  BOTTLE-FED  INFANTS  BETWEEN  1  AND  2  YEARS  OF  AGE  WITH  MILK 
FROM  DIFFERENT  SOURCES,  AND  THE  NUMBER  OF  BACTERIA  PRESENT  IN  THE 
MILK. 


Infants  under  one  year. 

Infanta  over  one  year. 

~, 

>» 

Diarrhoea. 

o*- 

fc^ 

Diarrhoea. 

Character  of  the  milk. 

\l 

r< 

-    — 
•'    -      — 

ill 

* 

3 

i 

Severe. 

1 

t*  *5       en^S 

II  III 

X        < 

2 
m 

I 

! 

1.  Pure  milk  boiled  and  modified  at] 

dispensary  or  stations  :  given  out 
in   small   bottles.     Milk   before  }• 

.41 

3  oz. 

10 

8 

11 

boiling  averaged  20,000  bacteria  | 

per  c.c.  ;  after  boiling  2  per  c.c.    J 

2.  Pure  milk,  24  hours  old,  sent  in] 

quart  bottles  to  tenements,  heated 
and  modified  at  home,  20,000  to 

23 

4J€" 

8 

5 

0 

24 

4%  oz. 

8 

2 

o 

200,000   bacteria   per  c.c.  when 

delivered.                                      J 

3.  Ordinary  milk,  36  hours  old.  from  ] 

a  selected  group  of  farms,  kept  | 

cool  in  cans  during  transport  ;  ! 
1,000,000  to  25,000,000  bacteria  per  f 
c.c.,  heated  and  modified  at  home 

18 

4      " 

6 

6 

i- 

12 

4      " 

1 

2         0 

before  using. 

4.  Cheap  milk,  36  to  60  hours  old, 

from   various  small   stores,  de- 

rived from  various  farms,  some 
fairlv   clean,  some  very   dirtv; 

21 

X" 

4 

13 

43 

7 

H  " 

1 

3         0 

400,000  to  175,000,000  bacteria  per 

c.c.                                              J 

5.  Condensed     milk     of    different] 

brands.  Made  up  with  hot  water.  ! 
As    given,    contained    bacteria  f 

9 

X" 

5 

2 

3 

4 

3%  " 

1 

3 

0 

from  5000  to  200,000  per  c.c. 

6.  Breast  milk      

16 

zy4" 

5 

2 

0 

There  is  nothing  in  the  observations  to  show  that  fairly  fresh  milk 
from  healthy  cows,  living  under  good  hygienic  conditions  and  con- 
taining, on  some  days,  when  delivered,  as  many  as  200,000  bacteria 
per  cubic  centimetre,  had  any  bacteria  or  any  products  due  to  bacteria 
that  remained  deleterious  after  the  milk  was  heated  to  near  the  boiling 
point. 

On  the  other  hand,  it  is  possible  that  certain  varieties  of  bacteria 
may,  under  conditions  that  are  unsanitary,  find  entrance  to  milk  and 
survive  moderate  heat  or  may  develop  poisonous  products  resistant 
to  heat  in  sufficient  amount  to  be  harmful,  even  when  they  have  accu- 
mulated to  less  than  200,000  per  cubic  centimetre. 

Turning  now  to  the  results  of  feeding  with  milk  which  has  been 
heated  and  which  before  sterilization  contained  from  1,000,000  to 


1  This  infant  died  from  enteritis  and  toxaemia. 

2  This  infant  died  of  pneumonia.    There  had  been  no  severe  intestinal  disorder  noted. 

3  One  of  the  four  had  pertussis,  the  remaining  three  died  from  uncomplicated  enteritis. 


460  BACTERIA  PATHOGENIC  TO  MAN 

25,000,000  bacteria  per  cubic  centimetre,  averaging  about  15,000,000, 
though  obtained  from  healthy  cows  living  under  fairly  decent  conditions 
and  although  the  milk  was  kept  moderately  cool  in  transit,  we  find  a 
distinct  increase  in  the  amount  of  diarrhceal  diseases.  Though  it  is 
probable  that  the  excessive  amount  of  diarrhoea  in  this  group  of  children 
was  due  to  bacterial  changes  which  were  not  neutralized  by  heat  or  to 
living  bacteria  which  were  not  killed,  yet  it  is  only  fair  to  consider  that 
the  difference  was  not  very  great  and  that  the  infants  of  this  group  were 
under  surroundings  not  quite  as  good  as  those  on  the  purer  milk. 

Finally,  we  come  in  this  comparison  to  the  infants  who  received 
the  cheap  store  milk  Pasteurized.  This  milk  had  frequently  to  be 
returned  because  it  curdled  when  boiled,  arid  contained,  according  to 
the  weather,  from  4,000,000  to  200,000,000  bacteria  per  cubic  centimetre. 
In  these  infants  the  worst  results  were  seen.  This  is  shown  not  only 
by  the  death  rate,  but  by  the  amount  and  the  severity  of  the  diarrhoeal 
diseases,  and  the  general  appearance  of  the  children  as  noted  by  the  phy- 
sicians. Although  the  average  number  of  bacteria  in  the  milk  received 
by  this  group  is  higher  than  that  received  by  the  previous  group,  the 
difference  in  results  between  this  group  and  the  previous  one  can  hardly 
be  explained  by  the  difference  in  the  number  of  bacteria.  The  varieties 
of  bacteria  met  with  in  this  milk  were  more  numerous  than  in  the  better 
milk,  but  we  were  unable  to  prove  that  they  were  more  dangerous. 
Probably  the  higher  temperature  at  which  the  milk  was  kept  in  transit 
and  the  longer  interval  between  milking  and  its  use  allowed  more  toxic 
bacterial  products  to  accumulate. 

Summary. — The  observations  here  recorded  were  made  upon  the 
groups  of  infants  for  periods  of  about  three  months  only,  and  the  con- 
clusions1 drawn  relate  especially  to  the  more  immediate  effects  of  the 
milk: 

1.  During  cool    weather    neither  the    mortality  nor  the    health   of 
the  infants  observed  in  the  investigation  was  appreciably  affected  by 
the  kind  of  milk  or  by  the  number  of  bacteria  which  it  contained.    The 
different  grades  of  milk  varied  much  less  in  the  amount  of  bacterial 
contamination  in  winter  than  in  summer,  the  store  milk  averaging 
only  about  750,000  bacteria  per  cubic  centimetre. 

2.  During  hot  weather  when   the   resistance   of  the   children   was 
lowered,  the  kind  of  milk  taken  influenced  both  the  amount  of  illness 
and  the  mortality;  those  who  took  condensed  milk  and  cheap  store 
milk  did  the  worst,  and  those  who  received  breast  milk,  pure  bottled 
milk,  and  modified  milk  did  the  best.    The  effect  of  bacterial  contami- 
nation was  very  marked  when  the  milk  was  taken  without  previous 
heating;  but,  unless  the  contamination  was  very  excessive,  only  slight 
when  heating  was  employed  shortly  before  feeding. 

3.  The   number  of  bacteria   which   may   accumulate   before   milk 
becomes  noticeably  harmful  to  the  average  infant  in  summer  differs 
with  the  nature  of  the  bacteria  present,  the  age  of  the  milk,  and  the 

1  These  conclusions  were  drawn  up  by  the  writer  in  association  with  Dr.  L.  E.  Holt,  after  a  joint 
study  of  the  results  obtained  in  the  studies  above  recorded. 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     461 

temperature  at  which  it  has  been  kept.  When  milk  is  taken  raw,  the 
fewer  the  bacteria  present  the  better  are  the  results.  Of  the  usual 
varieties,  over  1,000,000  bacteria  per  cubic  centimetre  are  certainly  dele- 
terious to  the  average  infant.  However,  many  infants  take  such  milk 
without  apparently  harmful  results.  Heat  above  170°  F.  (77°  C.)  not 
only  destroys  most  of  the  bacteria  present,  but,  apparently,  some  of  their 
poisonous  products.  No  harm  from  the  bacteria  previously  existing 
in  recently  heated  milk  was  noticed  in  these  observations  unless  they 
had  amounted  to  many  millions,  but  in  such  numbers  they  were  decid- 
edly deleterious. 

4.  AYhen  milk  of  average  quality  was  fed  sterilized  and  raw,  those 
infants  who  received  milk  previously  heated  did,  on  the  average,  much 
better  in  warm  weather  than  those  who  received  it  raw.     The  differ- 
ence was  so  quickly  manifest  and  so  marked  that  there  could  be  no 
mistaking  the  meaning  of  the  results.      The  bacterial  content  of  the 
milk  used  in  the  test  was  somewhat  less  than  in  the  average  milk  of 
the  city. 

5.  No  special  varieties  of  bacteria  were  found  in  unheated  milk  which 
seemed  to  have  any  special  importance  in  relation  to  the  summer  diar- 
rhoeas of  children.    The  number  of  varieties  was  very  great,  and  the  kinds 
of  bacteria  differed  according  to  the  locality  from  which  the  milk  came. 
None  of  the  139  varieties  selected  as  most  distinct  among  those  obtained 
injured  very  young  kittens  when  fed  in  pure  cultures.      A  few  cases  of 
acute  indigestion  were  seen  immediately  following  the  use  of  Pasteur- 
ized milk  more  than  thirty-six  hours  old.    Samples  of  such  milk  were 
found  to  contain  more  than  100,000,000  bacteria  per  cubic  centimetre, 
mostly  spore-bearing  varieties.    The  deleterious  effects,  though  striking, 
were  neither  serious  nor  lasting. 

At  the  present  time  there  is  in  New  York  City  no  general  sale  from 
stores  of  " Pasteurized"  •  or  "sterilized"  milk;  so  that  here  it  is  very 
rare  for  such  milk  to  be  used  thirty-six  hours  after  heating. 

6.  After  the  first  twelve  months  of  life,  infants  are  less  and  less  affected 
by  the  bacteria  in  milk  derived  from  healthy  cattle.    According  to  these 
observations,  when  the  milk  had  been  kept  cool  the  bacteria  did  not 
appear  to  injure  the  children  over  three  years  of  age,  at  any  season  of 
the  year,  unless  in  very  great  excess. 

7.  Since  a  large  part  of  the  tenement  population  must  purchase  its 
milk  from  small  dealers,  at  a  low  price,  everything  possible  should  be 
done  by  health  boards  to  improve  the  character  of  the  general  milk 
supply  of  cities  by  enforcing  proper  legal  restrictions  regarding  its 
transportation,   delivery,   and   sale.     Sufficient   improvements   in   this 
respect  are  entirely  feasible  in  every  large  city  to  secure  to  all  a  milk 
which  will  be  wholesome  after  heating.    The  general  practice  of  heat- 
ing milk,  which  has  now  become  a  custom  among  the  tenement  popula- 
tion of  New  York,  is  undoubtedly  a  large  factor  in  the  lessened  infant 
mortality  during  the  hot  months. 

8.  Of  the  methods  of  feeding  now  in  vogue  that  by  milk  from  central 
distributing  stations   unquestionably  possesses   the   most  advantages, 


462  BACTERIA  PATHOGENIC  TO  MAN 

in  that  it  secures  some  constant  oversight  of  the  child,  and,  since  it 
furnishes  the  food  in  such  a  form  that  it  leaves  the  mother  least  to  do, 
it  gives  her  the  smallest  opportunity  of  going  wrong.  This  method  of 
feeding  is  one  which  deserves  to  be  much  more  extensively  employed, 
and  might,  in  the  absence  of  private  philanthropy,  wisely  be  under- 
taken by  municipalities  and  continued  for  the  four  months  from  May 
15th  to  September  15th. 

9.  The  use,  for  infants,  of  milk  delivered  in  sealed  bottles,  should 
be  encouraged  whenever  this  is  possible,  and  its  advantages  duly  ex- 
plained.   Only  the  purest  milk  should  be  taken  raw,  especially  in  sum- 
mer. 

10.  Since  what  is  needed  most  is  intelligent  care,  all  possible  means 
should  be  employed  to  educate  mothers  and  those  caring  for  infants 
in  proper  methods.    This,  it  is  believed,  can  most  effectively  be  done  by 
the  visits  of  properly  qualified  trained  nurses  or  women  physicians  to 
the  homes,  supplemented  by  the  use  of  printed  directions. 

11.  Bad  surroundings,  though  contributing  to  bad  results  in  feed- 
ing, are  not  the  chief   factors.      It  is  not,  therefore,  merely  by  better 
housing  of  the  poor  in  large  cities  that  we  will  see  a  great  reduction  in 
infant  mortality. 

12.  While  it  is  true  that  even  in  tenements  the  results  with  the  best 
bottle  feeding  are  nearly  as  good  as  average  breast  feeding,  it  is  also 
true  that  most  of  the  bottle  feeding  is  at  present  very  badly  done;  so 
that,  as  a  rule,  the  immense  superiority  of  breast  feeding  obtains.    This 
should,  therefore,  be  encouraged  by  every  means,  and  not  discontinued 
without  good  and  sufficient  reasons.     The  time  and  money  required 
for  artificial  feeding,  if  expended  by  the  tenement  mother  to  secure  better 
food  and  more  rest  for  herself,  would  often  enable  her  to  continue 
nursing  with  advantage  to  her  child. 

13.  The  injurious  effects  of  table  food  to  infants  under  a  year  old, 
and  of  fruits  to  all  infants  and  young  children  in  cities,  in  hot  weather, 
should  be  much  more  generally  appreciated. 

Influence  of  Temperature  upon  the  Multiplication  of  Bacteria  in  Milk.— 
Few,  even  of  the  well  informed,  appreciate  how  great  a  difference  a  few 
degrees  of  temperature  will  make  in  the  rate  of  bacterial  multiplication. 
Milk  rapidly  and  sufficiently  cooled  keeps  almost  unaltered  for  thirty- 
six  hours,  while  milk  insufficiently  cooled  deteriorates  rapidly. 

The  majority  of  the  bacteria  met  with  in  milk  grow  best  at  tempera- 
tures above  70°  F.,  but  they  also  multiply  slowly  even  at  40°  F.;  thus 
of  60  species  isolated  by  us,  42  developed  good  growths  at  the  end  of 
seven  days  at  39°  F.  Our  observations  have  shown  that  the  bacteria 
slowly  increase  in  numbers  after  the  germicidal  properties  of  the  milk 
have  disappeared,  and  the  germs  have  become  accustomed  to  the  low 
temperature.  In  fact,  milk  cannot  be  permanently  preserved  unaltered 
unless  kept  at  32°  F.  or  less.  The  degree  of  cooling  to  which  ordinary 
supplies  of  milk  are  subjected  differs  greatly  in  various  localities. 
Some  farmers  chill  their  milk  rapidly,  by  means  of  pipe-coils  over 
which  the  milk  flows;  others  use  deep  wooden  tanks  rilled  with  water 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     463 

into  which  the  cans  of  milk  are  placed  soon  after  milking.  In  winter 
these  methods  are  very  satisfactory,  for  the  water  runs  into  the  pipes 
or  tanks  at  about  38°  F.  In  warmer  weather  they  are  unsatisfactory, 
unless  ice  is  used,  as  the  natural  temperature  of  the  water  may  be.  as 
high  as  55°  F.  A  considerable  quantity  of  milk  is  not  cooled  at  all  at 
the  farms.  It  is  sent  to  the  creamery  or  railroad  after  two  to  six  hours, 
and  is  then  more  or  less  cooled.  These  few  hours  in  summer,  when 
the  milk  is  left  almost  at  blood  heat,  allow  an  enormous  development 
of  bacteria  to  take  place,  as  is  shown  in  the  table  below. 

TABLE  I. — Showing  the  development  of  bacteria  in  two  samples  of  milk  maintained 
at  different  temperatures  for  twenty-four,  forty-eight,  and  ninety-six  hours,  respectively. 
The  first  sample  of  milk  was  obtained  under  the  best  conditions  possible,  the  secoud  in 
the  usual  way.  When  received,  specimen  No.  1  contained  3000  bacteria  per  c.c.,  speci- 
men No.  2,  30,000  per  c.c. 

Time  which  elapsed  before  making  test. 


Temperature. 
Fahrenheit. 


32° 


39° 


42° 


46C 


50° 


55C 


60° 


68C 


86C 


48hrs. 

2100 

27,000 

3600 

56,000 

3600 

210,000 

12,000 

360,000 

540,000 

1,940,000 

3,400,000 

38,000,000 

28,000,000 

168,000,000 


96hrs. 

1850 

24,000 

218,000 

4,300,000 

500,000 

5,760,000 

1,480,000 

12,200,000 

300,000,000 

1,200,000,000 


168  hrs. 

1400 

19,000 

4,209,000 

38,000,000 

11,200,000 

120,000,000 

80,000,000 

300,000,000 


24hrs. 
2400 
30,000' 
2500 
38,000 
2600 
43,000 
3100 
42,000 
11,600 
89,000 
18,800 
187,000 
180,000 
900,000 
450,000 
4,000,000 
1,400,000,000 
14,000,000,000 

Observations  on  Bacterial  Multiplication  in  Milk  at  90°  F.,  a  Temperature  Common 
in  New  York  in  Hot  Summer  Weather. 


Milk  I. 

Fresh  and  of  good 
quality. 

Original  number 

After  two  hours 

"     four     " 

"     six       " 

"     eight    " 


TABLE  II. — Number  of  Bacteria  per  1 

Milk  II. 

Fair  quality  from 
store. 

5200 

8400 
12,400 
68,500 


654,000 


92,000 
184,000 
470,000 
1,260,000 
6,800,000 


Milk  III. 
Bad  quality  from 
store. 

2,600,000 

4,220,000 

19,000,000 

39,000,000 

124,000,000 


A  sample  of  milk  No.  I.  removed  after  six  hours  and  cooled  to  50°  F.  contained 
145,000,000  at  the  end  of  twenty-four  hours.  Some  of  this  milk,  kept  cool  from  the 
beginning,  contained  but  12,800  bacteria  per  c.c.  at  the  end  of  twenty-four  hours. 


The  figures  referring  to  tests  of  the  second  sample  are  printed  in  heavy-face  type. 


BACTERIA  PATHOGENIC  TO  MAN', 

1.  The  number  of  bacteria  present  at  the  time  of  milking  and  twenty-four,  forty- 
eight,  and  seventy-two  hours  afterward  in  milk  obtained  and  kept  under  correct  con- 
ditions. 

No  preservatives  were  present  in  any  of  the  following  specimens: 

Number  of  Bacteria  in  Pure  Commercial  Milk. 

TABLE  III.— Milk  obtained  where  every  reasonable  means  was  taken  to  ensure  clean- 
liness. The  long  hairs  on  the  udder  were  clipped  ;  the  cows  roughly  cleaned  and 
placed  in  clean  barns  before  milking  ;  the  udders  were  wiped  off  just  previous  to  milk- 
ing ;  the  hands  of  the  men  were  washed  and  dried ;  the  pails  used  had  small  (six-inch ) 
openings,  and  were  thoroughly  cleaned  and  sterilized  by  steam  before  use.  Milk  cooled 
within  one  hour  after  milking  to  45°  R,  and  subsequently  kept  at  that  temperature. 
The  first  six  specimens  were  obtained  from  individual  cows  ;  the  last  six  from  mixed 
milk  as  it  flowed  at  different  times  from  the  cooler.  Temperature  of  barns  55°  F. 

Number  of  Bacteria  in  1  c.c.  of  Milk.1 

From  six  individual  cows. 
5  hrs 
after  milking.      After  24  hrs.       After  48  hrs.  After  72  hrs. 

500  700  12,500  Not  counted. 

700  700  29,400 

19,900  5200  24,200                   " 

400  200  8,600                   "         " 

900  1600  12,700                    "         " 

13,600  3200  19,500 

Average           6000  1933  17,816 

From  mixed  milk  of  entire  herd. 

6900  12,000  19,800  494,000 

6100  2,200  20,200  550,000 

4100  700  7,900  361,000 

1200  400  7,100  355,000 

6000  900  9,800  445,000 

1700  400  8,700  389,000 


Average  4333  2766  10,583  329,000 

Twenty-five  samples  taken  separately  from  individual  cows  on  another  day  and  tested 
immediately  averaged  4550  bacteria  per  c.c.  and  4500  after  twenty-four  hours.  These 
twenty -five  specimens  were  kept  at  between  45°  and  50°  F. 

2.  Milk  taken  during  winter  in  well-ventilated,  fairly  clean,  but  dusty  barns. 
Visible  dirt  was  cleaned  off  the  hair  about  the  udder  before  milking.  Milkers'  hands 
were  wiped  off,  but  not  washed.  Milk  pails  and  cans  were  clean,  but  the  straining  cloths 
dusty.  Milk  cooled  within  two  hours  after  milking  to  45°  F. 

1  Number  of  bacteria  obtained  from  development  ot  colonies  in  nutrient  agar  in  Petri  plates.  The 
nutrient  medium  contained  2  percent,  peptone  and  1.2  per  cent,  agar,  and  was  faintly  alkaline  to 
litmus.  One  set  of  plates  were  usually  left  four  days  at  about  20°  C.,  and  one  set  twenty-four  hours 
at  37°  C.,  and  then  twenty-four  hours  at  20°  C.  From  5  to  30  per  cent,  more  colonies  developed  as  a 
rule  in  the  plates  kept  at  room  temperature  than  in  those  kept  for  twenty-four  hours  at  37°  C.  The 
milk  was  diluted  as  desired  with  100  or  10,000  parts  of  sterile  water,  and  1  c.c.  of  the  diluted  milk 
was  added  to  8  c.c.  of  melted  nutrient  agar.  Plates  containing  over  1000  colonies  were  found  to  be 
inaccurate,  in  that  they  gave  too  low  totals.  Apparently  a  considerable  number  of  bacteria  failed 
to  develop  colonies  when  too  many  were  added  to  the  nutrient  agar.  Nutrient  gelatin  was  found  to 
be  more  troublesome  and  not  to  yield  more  accurate  results  than  nutrient  agar. 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     465 

TABLE  IV. — Number  of  Bacteria  in  1  c.c.  of  Milk. 
At  time  of  milking.                After  24  hrs.  Aft^er  48  hrs. 

12,000  14,000  57,000 

13,000  20,000  65,000 

21,500  31,000  106,000 


Average  15,500  21,666  76,000 

y  umber  in  City  Milk. 

3.  The  condition  of  the  average  city  milk  is  very  different,  and  is  shown  in  the 
following  tables. 

The  twenty  samples  were  taken  late  in  March  by  Inspectors  of  the  Department  of 
Health  of  New  York  City  from  cans  of  milk  immediately  upon  their  arrival  in  the  city. 
The  temperature  of  the  atmosphere  averaged  50°  F.  during  the  previous  twenty-four 
hours.  The  temperature  of  the  milk  when  taken  from  the  cans  averaged  45°  F.  Much 
of  this  milk  had  been  carried  over  two  hundred  miles.  From  the  time  of  its  removal 
from  the  cans,  which  was  about  2  A.M.,  until  its  dilution  in  nutrient  agar,  at  10  A.M., 
the  milk  was  kept  at  about  45°  F. 

TABLE  V. 
From  New  York  and  Hudson  River  Railroad.  From  Harlem  Railroad. 

No.  of  bacteria  No.  of  bacteria 

No.  of  sample.  in  1  c.c.  No.  of  sample.  in  1  c.c. 

50  ...  35,200,000  48  ...  6,200,000 

51  ...  13,000,000  49  ...  2,200,000 

52  ...  2,500,000  50  ...  15,000,000 

53  ...  1,400,000  51  ...  70,000 

54  ...  200,000  52  ...  80,000 

55  ...  600,000  53  ...  320,000 

While  the  above  figures  indicate  that  much  of  the  milk  sold  is  fair, 
even  in  summer,  they  show  an  appalling  condition  for  most  of  that  sold 
to  the  poorer  classes — those  who  not  only  comprise  the  larger  part  of 
the  population,  but  who  are  also  compelled  to  keep  their  children  in 
town  cluring  the  hot  weather. 

It  must  be  kept  in  mind  that  milk  averaging  3,000,000  bacteria  per 
cubic  centimetre  will,  when  kept  at  the  temperature  common  in  the 
homes  of  the  poor,  soon  contain  very  largely  increased  numbers  and 
show  its  dangerous  condition  by  turning  sour  and  curdling. 

Cleanliness  Used  in  Obtaining  Milk,  and  its  Influence. — The  present 
conditions  under  which  much  of  the  milk  is  obtained  are  not  pleasant 
to  consider.  In  winter,  and  to  a  less  extent  at  other  seasons  of  the  year, 
the  cows  in  many  stables  stand  or  lie  down  in  stalls  in  the  rear  portion 
of  which  there  is  from  one  to  four  inches  of  manure  and  urine.  When 
milked  the  hands  of  the  milkers  are  not  cleansed,  nor  are  the  under 
portions  of  the  cows,  only  visible  masses  of  manure  adhering  to  the 
hair  about  the  udder  being  removed.  Some  milkers  even  moisten 
their  hands  with  milk,  to  lessen  friction,  and  thus  wash  off  the  dirt  of 
their  hands  and  of  the  cow's  teats  into  the  milk  in  the  pails.  Some 
may  regard  it  as  an  unnecessary  refinement  to  ask  that  farmers  should 
roughly  clean  the  floors  of  their  stalls  once  each  day,  that  no  sweeping 
should  be  done  just  before  milking,  and  that  the  udders  should  be 

30 


466  BACTERIA  PATHOGENIC  TO  MAN 

wiped  with  a  clean  damp  cloth  and  the  milkers  should  thoroughly 
wash  and  wipe  their  hands  before  commencing  milking.  The  pails 
and  cans  should  not  only  be  carefully  cleansed,  but  afterward  scalded 
out  with  boiling  water.  The  washing  of  the  hands  would  lessen  the 
number  of  ordinary  filth  bacteria  in  the  milk,  and  diminish  risk  of 
transmitting  to  milk  human  infectious  diseases  like  scarlet  fever,  diph- 
theria, and  enteric  fever,  by  the  direct  washing  off  of  the  disease  germs 
from  infected  hands.  It  would  also  inculcate  general  ideas  of  the  neces- 
sity of  cleanliness  and  of  the  danger  of  transmitting  disease  through 
milk.  The  value  of  cleanliness  in  limiting  the  number  of  bacteria  is 
demonstrated  by  the  figures  contained  in  the  tables. 

Summary  and  Conclusions. — Because  of  its  location  and  its  hairy 
covering  the  cow's  udder  is  always  more  or  less  soiled  with  dirt  and 
manure  unless  cleaned.  On  account  of  the  position  of  the  pail  and  the 
access  of  dust-laden  air  it  is  impossible  to  obtain  milk  by  the  usual 
methods  without  mingling  with  it  a  considerable  number  of  bacteria. 
With  suitable  cleanliness,  however,  the  number  is  far  less  than  when 
filthy  methods  are  used,  there  being  no  reason  why  fresh  milk  should 
contain  in  each  cubic  centimetre  on  the  average,  more  than  12,000 
bacteria  per  c.c.  in  warm  weather  and  5000  in  cold  weather.  Such  milk, 
if  quickly  cooled  to  46°  F.,  and  kept  at  that  temperature,  will  at  the  end 
of  thirty-six  hours  contain  on  the  average  less  than  50,000  bacteria  per 
cubic  centimetre,  and  if  cooled  to  40°  F.  will  average  less  than  its 
original  number. 

With  only  moderate  cleanliness  such  as  can  be  employed  by  any 
farmer  without  adding  appreciably  to  his  expense,  namely,  clean  pails, 
straining  cloths,  cans  or  bottles,  and  hands,  a  fairly  clean  place  for 
milking,  and  a  decent  condition  of  the  cow's  udder  and  the  adjacent 
belly,  milk  when  first  drawn  will  not  average  in  hot  weather  over  30,000, 
and  in  cold  weather  not  over  25,000  bacteria  per  cubic  centimetre. 
Such  milk,  if  cooled  to  and  kept  at  50°  F.,  will  not  contain  at  the  end 
of  twenty-four  hours  over  100,000  bacteria  per  cubic  centimetre.  If 
kept  at  40°  F.  the  number  of  bacteria  will  not  be  over  100,000  per 
cubic  centimetre  after  forty-eight  hours. 

If,  however,  the  hands,  cattle,  and  barns  are  filthy,  and  the  pails  are 
not  clean,  the  milk  obtained  under  these  conditions  will,  when  taken 
from  the  pail,  contain  very  large  numbers  of  bacteria,  even  up  to  a 
million  or  more  per  cubic  centimetre. 

Freshly  drawn  milk  contains  a  slight  and  variable  amount  of  bac- 
tericidal substances  which  are  capable  of  inhibiting  bacterial  growth. 
At  temperatures  under  50°  F.  these  substances  act  efficiently  (unless  the 
milk  is  filthy)  for  from  twelve  to  twenty-four  hours,  but  at  higher  tem- 
peratures their  effect  is  very  soon  completely  exhausted,  and  the  bac- 
teria in  such  milk  will  then  rapidly  increase.  Thus  the  bacteria  in  fresh 
milk  which  originally  numbered  5000  per  cubic  centimetre  decreased 
to  2400  in  the  portion  kept  at  42°  F.  for  twenty-four  hours,  but  rose 
to  7000  in  that  kept  at  50°  F.,  to  280,000  in  that  kept  at  65°  F.,  and 
to  12,500,000,000  in  the  portion  kept  at  95°  F. 


BACTERIOLOGY  OF  MILK  IN  ITS  RELATION  TO  DISEASE     467 

As  we  have  seen,  the  milk  in  New  York  City  is  found  on  bacterio- 
logical examination  to  contain,  as  a  rule,  excessive  numbers  of  bacteria. 
During  the  coldest  weather  the  milk  in  the  shops  averages  over  300,000 
bacteria  per  cubic  centimetre,  during  cool  weather  about  1,000,000, 
and  during  hot  weather  about  5,000,000.  The  milk  in  other  large 
cities  is,  from  all  accounts,  in  about  the  same  condition. 

The  above  statement  holds  for  milk  sold  at  the  ordinary  shops,  and 
not  that  of  the  best  of  the  special  dairies,  where,  as  previously  stated, 
the  milk  contains  only  from  10,000  to  30,000  bacteria,  according  to  the 
season  of  the  year. 

The  question  might  be  raised,  Are  even  these  enormous  numbers 
of  bacteria  in  milk  during  hot  weather  actually  harmful? 

Our  knowledge  is  probably  as  yet  insufficient  to  state  just  how  many 
bacteria  must  accumulate  to  make  them  noticeably  dangerous  in  milk. 
Some  varieties  are  undoubtedly  more  harmful  than  others  and  we 
have  no  way  of  restricting  the  kinds  that  will  fall  into  milk,  except  by 
enforcing  cleanliness.  We  have  also  to  consider  that  milk  is  not  entirely 
used  for  some  twelve  hours  after  being  purchased,  and  that  during 
all  this  time  bacteria  are  rapidly  multiplying,  especially  where,  as 
among  the  poor,  no  provision  for  cooling  it  is  made.  Slight  changes 
in  the  milk  which  to  one  child  would  be  harmless,  would  in  another 
produce  disturbances  which  might  lead  to  serious  disease.  A  safe 
conclusion  is  that  no  more  bacterial  contamination  should  be  allowed 
than  it  is  practicable  to  avoid.  Any  intelligent  farmer  can  use  sufficient 
cleanliness  and  apply  sufficient  cold,  with  almost  no  increase  in  expense, 
to  supply  milk  twenty-four  to  thirty-six  hours  old  which  will  not  con- 
tain in  each  cubic  centimetre  over  50,000  to  100,000  bacteria,  and  no 
milk  containing  more  bacteria  should  be  sold. 

The  most  deleterious  changes  which  occur  in  milk  during  its  trans- 
portation are  now  known  not  to  be  due  to  skimming  off  the  cream,  or 
to  the  addition  of  water,  but  to  the  changes  produced  in  the  milk 
by  multiplication  of  bacteria.  During  this  multiplication,  acids  and 
distinctly  poisonous  bacterial  products  are  added  to  the  milk,  to  such 
an  extent  that  much  of  it  has  become  distinctly  deleterious  to  infants 
and  invalids.  It  is  the  duty  of  health  authorities  to  prevent  the  sale 
of  milk  rendered  unfit  for  use  through  excessive  numbers  of  bacteria 
and  their  products. 

The  culture  tests  to  determine  the  number  of  bacteria  present  in 
any  sample  of  milk  require  at  least  forty-eight  hours ;  so  that  the  sale 
of  milk  found  impure  cannot  be  prevented.  It  will,  however,  be  the 
purpose  of  the  authorities  gradually  to  force  the  farmers  and  the  middle- 
men to  use  cleanliness,  cold,  and  dispatch  in  the  handling  of  their  milk, 
rather  than  to  prevent  the  use  of  the  small  amount  tested  on  any  one  day. 

If  the  milk  on  the  train  or  at  the  dealer's  were  found  to  contain  exces- 
sive numbers  of  bacteria,  the  farmers  would  be  cautioned  and  instructed 
to  carry  out  the  simple  necessary  rules,  which  would  be  furnished. 

Transmission  of  Contagious  Diseases  through  Milk. — No  farmer  or 
dairyman  should  allow  anyone  who  has  a  contagious  disease,  or  who 


468  BACTERIA  PATHOGENIC  TO  MAN 

has  been  in  contact  with  any  person  having  scarlet  fever,  typhoid 
fever,  measles,  diphtheria,  or  consumption,  to  have  access  to  the  cattle, 
or  to  have  any  connection  with  the  milk  or  milking,  or  with  the  milk 
utensils.  Epidemics  and  outbreaks  of  contagious  disease  are  often 
produced  through  the  infection  of  the  milk  in  this  way.  Every  year 
epidemics  occur  which  have  been  traced  to  milk  contaminated  by 
ignorant  or  careless  milkmen,  who  have  infected  their  milk  from  their 
dirty  hands  or  the  dirty  water,  or  in  other  careless  ways.  This,  of 
course,  is  entirely  unnecessary  and  can  be  prevented.  The  extent  of 
this  danger  may  be  judged  by  the  fact  that  two  years  ago  there  was 
published  in  one  of  the  medical  journals  a  report  upon  330  outbreaks 
of  epidemic  diseases  traced  to  milk;  195  of  these  were  epidemics  of 
typhoid  fever,  in  147  of  which  the  disease  prevailed  at  the  dairy  or 
farm;  in  67  it  was  due  to  contamination  of  well-water;  in  24,  employe's 
at  the  farm  were  acting  as  nurses,  and  in  10  they  w~ere  working  while 
still  sick.  There  were  99  epidemics  of  scarlet  fever,  in  68  of  which 
the  source  of  infection  was  traced  to  the  illness  of  persons  at  the  dairy; 
in  17  the  employe's  were  themselves  suffering  from  scarlet  fever,  and  in 
10  they  were  acting  as  nurses  to  scarlet  fever  patients.  In  other  cases 
the  mode  of  infection  was  through  the  storage  of  milk  near  infected 
rooms,  or  the  poison  was  brought  by  cans  or  bottles  from  patients' 
houses.  There  were  36  epidemics  of  diphtheria,  in  13  of  which  the 
disease  existed  at  the  farm  or  dairy. 


PART  III. 
PROTOZOA.1 


CHAPTEK  XXXVII. 

CLASSIFICATION  AND  GENERAL  CHARACTERISTICS. 

ALL  animal  forms  consisting  throughout  their  entire  life  of  a  single 
cell  or  of  a  colony  of  single  cells  are  called  protozoa. 

They  are  so  closely  related  to  the  protophyta,  or  lowest  plant  forms, 
on  the  one  side,  and  the  metaphyta,  or  many-celled  animals,  on  the  other, 
that  it  is  difficult  to  mark  out  a  sharp  line  of  distinction  between  'them. 

In  general,  it  may  be  said  that  each  cell  consists  of  protoplasm  which 
is  differentiated  into  nucleus  and  cytoplasm,  both  parts  showing  many 
variations  in  the  several  groups,  and  that  each  cell  undergoes  a  more 
or  less  complicated  life  cycle,  appearing  in  different  forms  at  different 
stages  of  development. 

Doflein,  in  Kolle  and  Wassermann,  gives  the  grouping  of  the  protozoa 
as  follows: 

PHYLUM:   PROTOZOA. 

I.  Subphylum:  Plasmodroma.    Doflein. 
I.  Class:  Rhizopoda.    v.  Siebold. 

I.  Order:  Amcebina.    Ehrenberg. 
II.  Order:  Heliozoa.    Haekel. 

III.  Order:  Radiolaria.    Johannes  Miiller. 

IV.  Order:  Foraminifera.     d'Orbigny. 
V.  Order :  Mycetozoa.    de  Bary. 

II.  Class:  Mastigophora.    Diesing. 

I.  Subclass:  Flagellata.    Cohn  em.  Butschli. 

I.  Order:  Protomonadina.     Blochmann. 
II.  Order:  Polymastigina.    Biitechli  and  Blochmann. 

III.  Order:  Englenoidina.     Klebs. 

IV.  Order :  Chromomonadina.    Blochmann. 
V.  Order:  Phytomonadina.     Blochmann. 

II.  Subclass:  Dinoflagellata.     Biitschli. 
I.  Order :  Adinida.    Bergh. 
II.  Order:  Dinifera.    Bergh. 
III.  Subclass:  Cystoflagellata. 

Appendix:  Trichonymphidae.    Leidy. 

1  The  following  general  authorities  on  protozoa  have  been  consulted  freely  in  this  work :    Doflein 
and  Prowazek,  in  Kolle  and  Wassermann  ;  Minchin,  in  Ray  Lankester ;  Calkins. 


470  PROTOZOA 

PHYLUM:  PROTOZOA. 
III.  Class:  Sporozoa.    Leuckart. 

I.  Subclass:  Telosporidia.    Schaudinn. 

I.  Order  :  Coccidiomorpha.     Doflein. 

I.  Suborder:  Coccidia.     Leuckart. 

II.  Suborder :  Hfemosporidia.     Danilewski  em.  Schaudinn. 
II.  Order:  Gregarinida.    Aime"  Schneider  em.  Doflein. 
I.  Suborder :  Eugregarinida.    Doflein. 
II.  Suborder  :  Amoebosporidia.   Aime*  Schneider. 
II.  Subclass:  Neosporidia.    Schaudinn. 

I.  Order:  Cnidosporidia.    Doflein. 

I.  Suborder:  Myxosporidia.     Butschli. 
II.  Suborder:  Microsporidia.     Balbiani. 

II.  Order  :  Sarcosporidia.    Balbiani. 

Appendix  :  Serumsporidia,  Haplosporidia,  Lymphosporidia. 
II.  Subphylum:  Ciliophora.    Doflein. 
I.  Class:  Ciliata. 

I.  Order:  Holotricha.    Stein. 

II.  Order:  Heterotricha.    Stein. 

III.  Order:  Oligotricha.     Butschli. 

IV.  Order:  Hypotricha.    Stein. 
V.  Order:  Peritricha.    Stein. 

II.  Class:  Suctoria.    Butschli. 

So  far  only  a  few  of  the  protozoa  have  been  shown  to  produce  dis- 
ease in  man;  in  the  lower  animals  the  number  of  pathogenic  forms  is 
slightly  larger,  but  the  great  majority  seem  to  dwell  as  harmless  para- 
sites in  the  bodies  of  their  hosts. 

The  groups  which  are  of  medical  interest  are  the  following:  The 
amoebina1  and  the  mycetozoa,  from  the  class  rhizopoda;  the  flagellata, 
from  the  class  mastigophora;  the  sporozoa  as  a  class,  and  one  order 
from  the  ciliata. 

The  Amoebina  (Fig.  144)  include  forms  composed  of  naked,  simply 
constructed  protoplasm  having  the  power  of  producing  variously  shaped 
pseudopodia,  or  protoplasmic  processes,  which  are  used  as  organs  of 
locomotion  and  of  nutrition.  They  possess  generally  one  nucleus  and 
a  contractile  vacuole. 

The  life  cycle  of  only  a  few  varieties  has  been  studied,  and  until  the 
full  cycle  of  development  of  any  so-called  amoeba  is  known  it  is  impos- 
sible to  say  whether  it  belongs  among  the  rhizopoda,  or  whether  it  is 
one  of  the  forms  of  development  of  another  group,  as  amoeboid  forms 
may  occur  at  some  time  in  the  life  history  of  all  groups.  According  to 
the  few  forms  studied,  the  amoebse  increase  by  (1)  simple  division  and 
by  (2)  encysting.  In  the  latter  case  the  nucleus  divides  into  many 
daughter-nuclei,  which  arrange  themselves  around  the  periphery,2  and, 
under  favorable  conditions,  the  protoplasm  divides  about  them  and 
young  amcebse  are  formed,  leaving  often  a  central  mass  of  protoplasm, 
or  "crystal  residuum"  ("Rest  Korper").  The  young  amoeba  then 
break  through  the  cyst  membrane  and  soon  assume  the  forms  of  the 

i  See  figures  under  amoeba  coli.  2  See  figures  under  amoeba  coli. 


CLASSIFICATION  AXD  GENERAL  CHARACTERISTICS         471 

adult  amoebae.  In  one  species,  after  division  of  the  nucleus  and  proto- 
plasm, the  daughter-cells  become  "swarm  spores"  with  two  flagella. 
A  certain  form  of  conjugation  has  been  observed  in  some  varieties. 

Amoeba-  are  found  mostly  in  standing  fresh  water  and  sea-water  and 
on  moist  vegetable  substances.  A  few  have  been  described  in  the  body 
fluids  of  the  higher  animals.  Those  so  far  known  to  be  parasitic  for 
man  are  amoeba  coli  and  a  few  closely  related  varieties  which  are 
described  below. 

FIG. 140 


A.  Cell  of  root  of  cabbage  infiltrated  with  plasmodiophora  amoebae.  The  amoebae  are  fusing,  form- 
ing plasmodia.  B.  Beginning  mitotic  division  of  the  amoebae.  The  nucleus  of  the  host  cell  beneath. 
(After  Nawaschin.) 

The  Mycetozoa  are  a  group  of  organisms  showing  characteristics  of 
both  plants  and  animals.  They  are,  therefore,  claimed  by  botanists  as 
well  as  zoologists.  They  have  possessed  some  interest  of  late  from  a 
medical  standpoint  because  one  variety,  the  plasmodiophora  brassicae 
Waronin  (Figs.  140  and  141),  which  is  an  intra cellular  parasite  of  mem- 
bers of  the  cruciferse,  producing  large  tumors  in  their  roots  ("fingers 
and  toes,"  "club-foot"),  has  been  shown  to  form  cell  inclusions 
somewhat  similar  to  some  of  the  cell  inclusions  seen  in  the  carci- 
nomata,  and  certain  investigators  have  thought  that  it  bears  a  re- 
lation to  the  etiology  of  carcinoma.  But  so  far  no  relationship  has 
been  proven.  The  plasmodiophora  brassicae,  when  inoculated  into 
experimental  animals,  produces  only  small  granulomata  which  finally 
disappear. 


472 


PROTOZOA 


The  Flagellata1  are  organisms  composed  of  naked,  variously  constructed 
protoplasm,  and  moving  by  means  of  one  or  more  long  flagella.  Some 
forms  also  show  amoeboid  movements.  The  number  and  characteristics 
of  the  flagella  vary.  Generally  there  is  a  principal  flagellum  directed  for- 
ward, and  there  are  from  one  to  several  flagella  directed  backward.  Often 
a  flagellum  seems  to  be  attached  throughout  part  of  its  length  to  the  body 
of  the  organism  by  a  wavy  or  undulating  membrane.  The  protoplasm  of 
the  flagella  appears  homogeneous,  except  for  the  fact  that  it  may  occasion- 
ally show  small  granules.  The  flagella  arise  from  small  granules,  the  "  fla- 
gella roots,"  which,  according  to  some  authors,  are  similar  to  centrosomes, 
and,  according  to  others,  micronuclei.  The  body  of  the  flagellata  is  gen- 
erally round  or  oval  and  more  or  less  motile.  Its  protoplasm  may  be  finely 
or  coarsely  granular,  sometimes  reticular,  and  may  contain  one  or  more 
vacuoles,  one  or  two  of  which,  situated  anteriorly,  may  be  contractile.  It 

FIG. 141 


Two  cells  infiltrated  with  spores  of  the  plasmodiophora  brassicee.    (Doflein.) 

may  also  contain  various  food  particles.  The  nucleus  situated  anteriorly 
varies  in  appearance  in  the  different  species.  It  is  usually  more  or  less 
granular,  with  a  central  body,  but  in  some  species  it  is  vesicular.  The 
flagellata  multiply  either  in  the  purely  motile  condition  or  after  encysting. 
In  the  first  case  the  division  is  generally  longitudinal.  In  the  second  case 
they  may  or  may  not  conjugate  before  they  encyst.  Then  division  forms 
occur  in  the  cyst  by  a  process  somewhat  similar  to  that  in  the  aincebse. 

Flagellates  are  found  in  foul  and  stagnant  water,  in  the  ocean, 
and  a  few  in  rivers  and  in  the  body  fluids  of  higher  animals.  The  fol- 
lowing species  which  are  described  below  have  been  reported  as  being 
parasitic  in  man:  trypanosoma,  cercomonas,  trichomonas,  lamblia 
intestinalis. 

The  sporozoa  are  a  group  of  exclusively  parasitic  protozoa  of  very 
widespread  occurrence,  living  in  the  cells,  tissues,  and  cavities  of  ani- 

1  See  Figs.  146  and  147,  under  trypanosoma  Lewisi. 


CLASSIFICATION  AND  GENERAL  CHARACTERISTICS         473 

mals  of  every  class.  Generally  they  are  harmless,  but  some  varieties 
may  produce  pathological  changes  and  even  fatal  diseases  severely 
epidemic. 

As  their  name  indicates,  they  are  all  characterized  by  reproduction 
through  spore  formation,  but  they  exhibit  the  utmost  diversity  of  struc- 
tural and  developmental  characteristics.  As  a  rule,  each  'species  is 
parasitic  on  one  kind  of  tissue  of  a  particular  species  of  host.  They 
are  generally  taken  into  the  system  in  the  spore  stage  either  (1)  with 
the  food  of  the  host,  (2)  by  the  bites  of  insects,  or  (3)  by  inhalation. 
The  spore  membranes  are  dissolved  by  the  fluids  of  the  host,  and  thus 
one  or  more  germs  or  sporozoites  are  set  free  to  bore  into  the  special  cells 
of  the  host.  Here  they  grow,  some  remaining  intracellular  permanently, 

FIG.  142 


Diagram  of  variations  in  life  cycle  of  flagellates :  1,  a  young  flagellate  ;  2,  adult  flagellate ;  3,  lon- 
gitudinal division  of  adult  free  form ;  4,  daughter  flagellate  ;  5,  encystation  ;  6-8,  division  into 
isogametes ;  x  and  z,  division  into  macrogametes  and  microgametes,  characteristic  for  some  forms ;  9, 
conjugation  of  the  isogametes ;  y,  conjugation  of  the  macrogametes  and  microgametes ;  10,  resting- 
stage— zygote ;  11-12,  division  into  young.  (After  Doflein.) 

others  only  in  the  young  stages.  The  latter  either  pass  different  phases 
of  their  more  or  less  complicated  life  history  in  different  parts  of  the  body 
of  one  and  the  same  host,  or  they  pass  some  phases  of  their  life  cycle  in 
the  cells  of  an  intermediate  host. 

The  sporozoa  vary  widely  in  size  as  well  as  in  other  characteristics. 
From  the  smallest,  several  of  which  can  be  contained  in  a  single  blood 
cell,  there  are  all  gradations  in  size  up  to  those  that  may  be  seen  by 
the  naked  eye  (Prospora  gigantea,  16  mm.). 

Besides  being  characterized  by  the  power  to  produce  more  or  less 
resisting  spores,  the  sporozoa  are  also  characterized  by  the  fact  that  as 
a  class  they  possess  none  of  the  special  organs  found  in  other  protozoa 
for  ingesting  or  digesting  solids.  Many  develop  flagella  or  show  amoeboid 


474 


PROTOZOA 


movement  during  certain  stages  of  their  life  cycle,  but  the  flagella  and 
pseudopodia  are  organs  of  locomotion  and  not  of  nutrition  except  in 


FIG.  143 


CLASSIFICATION  AND  GENERAL  CHARACTERISTICS         475 

so  far  as  they  increase  the  absorptive  surface  of  the  body,  as  food  is 
absorbed  by  diffusion.  Food  vacuoles  or  contractile  vacuoles  have  not 
been  found. 

The  life  cycle  of  a  typical  sporozoan  is  represented  after  Schaudinn 
in  Fig.  143. 

A  somewhat  similar  cycle  may  be  followed  in  the  study  of  the  coc- 
cidiuin  cuniculi  of  the  rabbit,  a  description  of  which  is  given  below.  The 
other  varieties  in  this  group,  which  are  parasitic  in  man,  or  which  are 
of  some  medical  interest,  and  which  are  described  below,  are  the 
following: 

Various  not  fully  studied  coccidia. 

Plasmodium  malariae  and  its  allies. 

Piroplasma  bigemmium  and  its  allies. 

Nosema  bombycis  and  nosema  lophii. 

The  Ciliata  (See  Fig.  159)  belong  to  the  most  complex  of  the  protozoa. 
They  possess  a  definite  entoplasm  containing  nuclei  and  food  vacuoles, 
and  a  definite  ectoplasm  containing  basal  granules  from  which  arise  the 
cilia  which  give  the  group  its  name.  They  have  organoid  structures 
which  receive  the  food,  and  some  have  definite  mouth  openings  indeed, 
and  definite  places  for  excreting  waste  products.  The  food  vacuoles  may 
contain  acid  or  alkaline  digestive  products.  The  nuclear  material  is 
differentiated  into  two  forms,  a  large  macronucleus  and  a  much  smaller 
micronucleus.  The  function  of  the  macronucleus  is  supposed  to  be 
vegetative,  and  that  of  the  micronucleus  reproductive.  The  macro- 
nucleus  varies  in  size  and  shape  and  is  completely  filled  with  an  alveolar 

DESCRIPTION  OF  FIG.  143. 

The  life  cycle  of  coccidium  Schubergl.  /  to  VII  represent  the  asexual  reproduction  or  schizogony, 
commencing  with  infection  of  an  epithelial  cell  by  a  merozoite  or  a  sporozoite;  the  merozoite  after 
stage  VII  may  start  again  at  stage  //,  as  indicated  by  the  arrows,  or  it  may  go  on  to  the  formation 
of  gametocytes  (IX  to  XII).  IX  to  XIV  represent  the  sexual  generation,  the  line  of  development 
becoming  split  into  two  lines— male  5  and  female  9,  culminating  in  the  highly  differentiated  gametes, 
which  conjugate  and  become  again  a  single  line,  shown  in  X/Vand  XV.  The  zygote  thus  formed  goes 
on  to  the  production  of  spores,  XVI  to  XX.  I  to  IV  represent  epithelial  cells  showing  penetration 
of  a  merozoite  or  a  sporozoite  and  its  change  into  a  schizout.  V,  the  nucleus  of  the  schizout  divid- 
ing. VI,  numerous  daughter-nuclei  in  the  schizout.  VII,  segmentation  of  the  schizout  into  numerous 
merozoites,  about  a  central  mass  of  residual  protoplasm,  which  in  this  figure  is  hidden  by  the  mero- 
zoites.  VIII,  merozoite  passing  to  reinfect  host  cell  and  repeat  the  process  of  schizogony.  IX,  X,  mero- 
zoites to  be  differentiated  into  male  and  female  gametocytes.  XIa  and  Xlla,  the  two  gametocytes 
within  a  host  cell;  the  microgametocyte  (5)  has  fine  granulations;  the  macrogametocyte  (9) 
has  coarse  granulations.  Xlb,  an  immature  female  gametocyte  within  a  host  cell.  XIc,  a  female 
gametocyte  undergoing  maturation,  still  in  the  host  cell.  XIII,  mature  macrogametocyte,  freed 
from  the  host  cell,  and  sending  a  cone  of  reception  toward  an  approaching  microgametocyte. 
Xllb,  a  full-grown  micro-gametocyte  within  a  host  cell.  In  XIIc  the  nucleus  of  the  microgametocyte 
has  divided  up  to  form  a  great  number  of  daughter-nuclei.  In  Xlld  the  nuclei  of  the  last  stage  have 
become  microgametes,  each  with  two  flagella.  Xlle,  represents  the  free  microgametes,  swimming  to 
find  a  macrogamete.  XIV,  the  zygote  (fertilized  macrogamete),  surrounded  by  a  tough  membrane  or 
oticyst,  which  allows  no  more  microgametes  to  enter,  and  containing  the  female  chromatin,  which  is 
taking  the  form  of  a  spindle,  and  the  male  chromatin  in  a  compact  lump.  XV,  the  chromatin  from 
these  two  sources  united  and  no  longer  distinguishable  as  male  and  female.  XVI,  the  nucleus  of  the 
zygote  dividing.  In  XVII  tour  daughter-nuclei  are  formed— the  nuclei  of  the  sporoblasts.  In  XVIII 
the  four  gporoblasts  become  distinct,  leaving  a  small  quantity  of  residual  protoplasm ;  each  sporo- 
blast  has  formed  a  membrane,  the  sporocyst.  In  XIX  within  each  sporocyst  two  sporozoites  have 
been  found  about  a  sporal  residuum.  In  XX,  the  sporozoites  becoming  free  by  bursting  the  sporo- 
cysts,  pass  out  through  an  aperture,  in  the  wall  of  the  oiicyst,  and  are  ready  to  enter  the  epithelial 
cells  of  the  host.  (From  Lang.) 


476  PROTOZOA 

chromatin.  The  micronucleus  also  varies  in  size  and  shape  but  is  vesic- 
ular in  structure,  with  the  chromatin  heaped  in  one  mass.  Division 
of  the  nuclei  takes  place  by  mitosis  in  the  case  of  the  micronuclei,  and 
by  amitosis,  as  a  rule,  in  the  case  of  the  macronuclei.  Under  unfavor- 
able conditions  the  ciliata  may  encyst. 

Conjugation  is  necessary  to  the  life  activity  of  these  organisms.  The 
phenomena  of  conjugation  in  the  ciliata  has  been  well  worked  out.  The 
micronuclei  play  the  most  important  part,  whereas  the  macronuclei 
simply  break  up  and  disappear  in  the  protoplasm. 

According  to  the  arrangement  of  the  cilia  the  ciliata  are  divided  into 
the  five  orders  given  in  the  general  classification.  Among  these  the 
second,  the  order  of  the  Heterotricha,  interests  us.  In  the  Hetero- 
tricha  the  cilia  are  uniform  over  most  of  the  body,  while  a  special- 
ized set  fused  into  a  series  of  firm  vibratory  plates  are  found  about 
the  mouth. 

Only  one  genus — the  Balantidium,  described  in  a  later  chapter — 
has  been  observed  in  man. 

A  description  is  also  given  of  various  protozoan-like  forms  found  in 
the  following  diseases :  smallpox,  cowpox,  and  related  diseases ;  scarlet 
fever,  measles,  kala-azar,  and  hydrophobia. 


CHAPTER   XXXVIII. 

». 

ORDER:  AMGEBINA. 
Amoeba  coli  (Losch). 

SEVERAL  varieties  of  amoebae  have  been  reported  as  occurring  in 
the  human  intestines,  but  as  the  full  cycle  of  no  one  of  them  has 
been  worked  out  it  is  possible  that  some  of  them  at  least  may  be 
stages  in  the  life  cycle  of  other  protozoa.  The  most  important  one 
among  these  varieties  is  that  observed  by  Lambl  in  1860,  and  more 
fully  described  by  Losch  in  1875  as  amoeba  coli.  Losch  found  it  in  the 
stools  of  dysenteric  patients,  and  he  succeeded  in  producing,  experi- 
mentally, superficial  ulce rations  in  the  large  intestines  of  dogs.  He 
therefore  claimed  that  this  organism  is  the  cause  of  dysentery. 

His  work  was  corroborated  by  many  observers,  the  organism  being 
also  found  pure  in  tropical  abscess  of  the  liver;  but  later  studies  have 
shown  that  this  parasite  does  not  play  as  important  a  part  in  dysentery 
as  was  thought.  It  has  been  found  that  in  many  cases  of  dysentery  the 
amoeba  coli  is  not  present,  that  the  same  or  similar  amoebae  are  present 
in  the  healthy  stools,  and  that  animal  experiments  are  not  satisfactory. 
These  facts,  together  with  those  brought  out  by  the  recent  work  of 
Shiga,  Kruse,  Flexner,  and  others,  on  bacillary  dysentery,  have  demon- 
strated that  there  are  at  least  two  forms  of  dysentery,  one  produced  by 
amoebae  and  the  other  by  bacilli,  and  that  in  the  former  case,  among  a 
number  of  harmless  varieties  of  intestinal  amoebae,  one  or  two  only  are 
of  clinical  importance.  According  to  Shiga  the  differential  diagnosis 
between  the  two  varieties  of  dysentery  is  as  follows: 

In  amoebic  dysentery  (1)  the  disease  is  generally  chronic;  (2)  no 
dysentery  bacilli  are  found  in  the  feces;  (3)  there  are  not  present  any 
severe  toxic  symptoms,  such  as  fever  (except  in  the  case  of  abscess  of 
the  liver),  weakness,  headache,  anorexia,  rapid  emaciation,  hemor- 
rhages, etc.;  (4)  abscess  of  the  liver  is  a  frequent  sequela;  (5)  the 
lesion  is  in  the  caecum  and  descending  colon ;  the  small  intestines  are 
not  affected. 

In  bacillary  dysentery  the  finding  of  the  bacilli  and  the  positive  results 
of  agglutination  tests,  together  with  the  clinical  symptoms  of  intoxica- 
tion, make  a  certain  diagnosis. 

At  present  we  may  group  the  pathogenic  niiKrba1  under  one  head, 
calling  the  groups  amoeba  coli;  though  Schaudinn,  in  his  recent  work  on 
the  amoebae  of  the  human  intestines,  has  given  quite  a  definite  descrip- 
tion of  two  varieties,  one  of  which  he  calls  entamoeba  coli  Losch,  which 
is  a  harmless  comensal  in  the  human  intestines,  and  the  other  entamoeba 


478 


PROTOZOA 


hystolytica  Schaudinn,  which  corresponds  with  the  form  studied  by 
Councilman  and  Lafleur  and  by  Jiirgens,  and  which  is  pathogenic, 
producing  the  true  amoebic  dysentery.  He  says  that  the  pathogenic 
variety  has  a  more  definite  ectoplasm  than  the  non-pathogenic;  further- 
more, that  its  nucleus  contains  less  chromatin,  making  it  more  difficult 
to  demonstrate,  it  has  a  more  varied  form  and  is  always  situated  eccen- 
trically. This  variety  increases  by  division  into  two  and  by  budding, 
while  the  non-pathogenic  variety  increases  by  a  regular  division  into 
eight  daughter  forms  (schizogony).  In  unfavorable  conditions  both 
varieties  may  produce  cysts. 

Morphology. — The  size  of  the  amoeba  coli  is  given  variously  by  dif- 
ferent authors  within  the  limits  of  7^  to  50/*,  more  commonly  12 p.  to  30//. 
In  a  state  of  rest  it  assumes  a  spherical  shape  which  appears  discoid 
under  the  microscope.  It  may  generally  be  distinguished  from  the  other 
cellular  elements  found  in  the  feces  by  its  pale-greenish  tint  and  by  its 
stronger  refraction  of  light.  The  outline  of  the  body  ordinarily  appears 
as  a  thin,  single,  dark  line.  The  two  portions  of  the  body,  the  inner,  or 


Amoeba  coli.    a.  Containing  red  blood  cells  (Romer).    6.  Mode  of  division  (Harris). 
c.  Cyst  with  eight  nuclei  (Grassi). 

entoplasm,  which  is  more  or  less  granular  and  of  a  darker  color,  and 
the  outer,  or  ectoplasm,  which  is  homogeneous  and  of  a  lighter  color, 
cannot  always  be  made  out  and  are  more  evident  in  the  motile  than  in 
the  resting  amoeba. 

The  entoplasm  constitutes  the  greater  portion  of  the  body  of  the 
amoeba.  In  the  smaller  forms  it  is  finely  granular,  and  may  show  no 
other  structure.  In  the  larger  forms  it  is  more  coarsely  granular  and 
may  contain,  in  varying  numbers,  bacteria,  starch  bodies,  cell  detritus, 
and  red  and  white  blood  cells,  in  various  stages  of  digestion.  One  to 
several  vacuoles  have  been  seen  in  the  entoplasm,  but  only  Dock  has 
spoken  of  their  infrequent  pulsation. 

The  ectoplasm  forms  a  hyaline  zone  of  variable  thickness  about  the 
entoplasm.  It  has  the  appearance,  under  the  microscope,  of  finely 
ground  glass  of  a  distinctly  pale-green  color.  It  seems  often  to  pass  out 
into  short,  irregular  pseudopods. 

The  nucleus  of  the  amoeba  is  2/Jt  to  IfJ.  in  diameter.  It  is  a  more 
or  less  spherical  vesicle-like  body  containing  a  dark,  chromatin  inner 


AMCEBINA  479 

body  which  is  connected  with  the  thick  nuclear  membrane  by  a  pro- 
toplasmic network. 

It  is  not  always  easy  to  detect  the  nucleus  in  fresh  or  motile  amoebae, 
but  under  certain  conditions  in  motionless  or  dead  amoebae  it  becomes 
evident.  It  may  be  easily  shown  by  appropriate  staining  reagents. 

Biological  Characters. — The  most  striking  and  characteristic  feature 
of  the  amoeba  is  its  motility.  This  may  consist  either  in  an  alteration 
of  its  shape  or  in  an  actual  change  of  place.  Both  of  these  phenomena 
are  produced  through  the  mechanism  of  pseudopodia.  These  latter 
are  rounded,  blunt,  and  homogeneous  processes  formed  by  the  more 
or  less  gradual  protrusion  of  a  portion  of  the  ectoplasm  at  some  part 
of  the  periphery  of  the  amoeba.  The  motion  is  at  times  quite  gradual 
and  continuous,  at  others  sudden  and  jerky.  The  progressive  move- 
ment, that  is,  actual  locomotion,  is  brought  about  by  the  protrusion  of 
pseudopodia,  and  into  these,  when  they  have  reached  a  certain  size, 
the  more  or  less  granular  and  vacuolated  entoplasm,  with  its  contents, 
flows  with  a  more  rapid  movement  than  that  by  which  the  pseudo- 
podia themselves  were  formed.  Locomotion  is  generally  observed  to 
take  place  in  the  direction  of  least  resistance,  a  group  of  cellular  ele- 
ments or  some  detritus  being  sufficient  to  divert  the  course  of  the  amoeba. 
The  amoeboid  movements  are  also  influenced  by  various  other  factors, 
particularly  by  variations  of  temperature.  They  are  most  active  at 
the  mean  temperature  of  the  human  body,  becoming  less  active  as  the 
temperature  falls  or  rises  above  this  mean,  and  indeed  they  become  quite 
motionless  in  a  temperature  lower  than  75°  F.  According  to  Boas,  amoebae 
remain  alive  outside  of  the  body  for  not  more  than  twenty-four  hours. 

Sexual  reproduction  has  been  described  by  Schaudinn  for  some  forms; 
but  the  usual  modes  of  multiplication  are  by  simple  division  and  by 
division  after  cyst  formation.  Attempts  to  cultivate  amoebae  outside  of 
the  body  have  been  unsuccessful  until  recently,  when  Musgrave  and 
Clegg  described  the  cultivation  of  pure  species  in  pure  cultures  of 
bacteria — the  "  pure  mixed  cultures  "  of  Frosch. 

Animal  Experiments. — It  is  evident  that  in  the  absence  of  artificially  pro- 
duced pure  cultures  of  amoebae,  inoculation  experiments  must  be  made 
with  material  such  as  dysenteric  stools  or  the  contents  of  hepatic  abscesses. 
In  a  few  cases  where  material  has  been  obtained  from  hepatic  abscesses 
which  have  been  found  to  contain  no  organisms  other  than  amoebae,  the 
inoculations  have  been  made  in  three  ways:  (1)  by  feeding  animals  with 
material  containing  the  amoebae;  (2)  by  inoculation  into  the  small  intes- 
tines after  a  preliminary  laparotomy;  and  (3)  by  rectal  injections  with  or 
without  suture  of  the  anal  orifice.  The  first  method  has  always  proved 
unsuccessful,  except  when  encysted  forms  were  present.  To  the  second 
method  the  objection  has  been  raised  that  the  manipulation  of  the  intes- 
tines and  the  use  of  antiseptic  solutions  during  the  course  of  the  opera- 
tion are  in  themselves  a  source  of  irritation  to  the  bowel  and  in  some 
cases  have  produced  an  enteritis.  The  third  method  has  given,  though 
not  in  every  case  tried,  positive  results  in  the  hands  of  Losch,  Kruse, 
Pasquale,  Jurgens,  and  others.  In  the  successful  cases  the  lesions  found 


480 


PROTOZOA 


were  reddening  and  swelling  of  the  intestinal  mucosa,  chiefly  of  the 
lower  half  of  the  large  bowel,  with  here  and  there  ecchymoses,  small 
superficial  areas  of  necrosis,  and  shallow  ulcerations.  The  mesenteric 
glands  and  the  solitary  lymphoid  follicles  were  often  swollen.  In  the 
blood-tinged  mucus  covering  the  mucous  membranes  amoebae  were 
found  in  greater  or  less  numbers.  Microscopic  examinations  showed 
that  the  necrosis  was  limited  as  a  rule  to  the  mucosa,  and  that  beneath 
it  the  submucosa  was  thickened  and  cedematous  and  its  vessels  engorged; 
there  was  also  small-celled  infiltration.  Amcebse  were  found  in  the 
borders  of  the  ulcers,  chiefly  in  the  follicles  of  Lieberkiihn;  in  the  base 
of  the  ulcers  they  rarely  penetrated  more  deeply  than  the  upper  layers 
of  the  submucosa.  With  the  amcebse  were  found  many  bacteria,  chiefly 
streptococci. 

Concerning  the  source  of  the  amcebse  and  the  mode  of  infection  little 
can  be  positively  stated.     It  is  reasonable  to  suppose,  however,  that 


FIG.  145 


Jfct, 


Jfn 


Jfn 


Leydenia  gemmipara  Schaudinn.  A,  single  amoeba ;  B,  plasmodia  and  budding ;  w,  nucleus ;  n', 
nucleus  dividing ;  cv,  contractile  vacuole  ;  v,  vacuole  ;  er,  red  blood  cell ;  Kn,  buds ;  Ka,  amoeba 
developed  from  bud. 

the  mouth  must  be  the  usual  path  of  infection,  and  that  the  amcebse 
in  all  probability  are  taken  in  with  the  drinking-water  and  with  un- 
cooked vegetables. 

Methods  of  Examination. — The  stools  must  be  examined  in  as  fresh 
a  condition  as  possible.  The  bloody  masses  of  mucus  and  the  material 
about  them  should  be  chosen  to  be  examined.  It  may  be  necessary  to 
thin  the  particles  examined  with  physiological  salt  solution.  The  warm 
stage  should  be  used  and  the  cover-glass  should  be  supported  by  small 
feet  of  sealing  wax.  The  collected  material  should  be  kept  at  body 
temperature  until  time  of  examination. 

For  permanent  preparations  Jager  especially  recommends  the  fol- 
lowing method :  For  a  fixative  a  concentrated  watery  solution  of  sub- 
limate 100  c.c.,  absolute  alcohol  50  c.c.,  acetic  acid  5  drops.  After 
a  few  minutes'  fixation  wash  carefully  with  iodine-alcohol,  then  stain 
with  Grenadier's  hsematoxylin  ten  minutes,  afterward  wash  with  water 


AM(EBINA  481 

until  no  more  blue  stain  comes  from  the  specimen,  then  stain  with  1 
per  cent,  eosin.  The  nuclei  of  the  leukocytes  are  stained  blue,  while 
those  of  the  amoebae  are  stained  red. 

Mallory  and  Wright  recommended  the  following  method  for  sections 
containing  amceba  coli:  1.  Harden  in  alcohol.  2.  Stain  sections  in  a 
saturated  aqueous  solution  of  thionin  three  to  five  minutes.  3.  Differ- 
entiate in  a  2  per  cent,  aqueous  solution  of  oxalic  acid  for  one-half  to 
one  minute.  4.  Wash  in  water.  5.  Dehydrate  in  alcohol.  6.  Clear 
in  oleum  origani  cretici.  7.  Wash  off  with  xylol.  8.  Xylol  balsam. 

Amoebae  in  Diseases  Other  than  Dysentery. — Kartulis  reported  finding 
a  large  motile  amoeba  (30/*  to  38,«)  in  an  abscess  of  the  lower  jaw  of  an 
Arabian.  Flexner  also  observed  a  similar  case  in  a  sixty-two-year-old 
man.  Baelz  found  a  very  large  amoeba  in  the  bloody  urine  and  in  the 
vagina  of  a  twenty-three-year-old  Japanese  who  was  suffering  from 
tuberculosis  of  the  lung.  Jiirgens,  Kartulis,  and  Posner  also  reported 
finding  similar  amoebae  in  cases  of  cystitis  and  bloody  urine. 

In  the  ascitic  fluid  of  a  man  who  had  carcinoma  of  the  stomach 
Leyden  found  motile  cellular  elements  which  Schaudinn  pronounced 
independent  organisms  belonging  to  the  rhizopoda  Leydenia  gem- 
mipara  (Schaudinn),  Fig.  145.  Similar  organisms  were  found  in  the 
ascitic  fluid  of  a  girl  who  had  an  abdominal  tumor.  The  organisms 
remained  motile  in  the  ascitic  fluid  seven  days  after  its  removal.  The 
organism  possesses  a  pulsating  vacuole  and  one  vesicular  nucleus;  it 
divides  directly  and  by  budding.  The  individuals  seem  readily  to  fuse 
(plastogamy).  The  pathological  significance  of  this  rhizopod  is  not 
clear. 


31 


CHAPTER  XXXIX. 

TRYPANOSOMA. 

Subclass:  Flagellata. 

Order:  Protomonadina. 

Genus:  Trypanosoma.1 

The  genus  trypanosoma  includes  blood  parasites  of  the  vertebrates 
distinguished  by  a  somewhat  long  body  more  or  less  spirally  twisted, 
one  to  several  flagella,  an  undulating  membrane,  one  nucleus,  and  a 
"flagellum  root."  The  chief  flagellum  is  directed  forward,  arising  from 
or  near  a  small,  more  or  less  rounded  structure,  the  "flagellum  root " 
(blepharoplast) ,  situated  near  the  posterior  end  of  the  organism.  The 
nature  of  the  "flagellum  root"  is  still  a  matter  of  controversy.  Some 
consider  it  of  the  nature  of  a  centrosome,  others,  of  that  of  a  micro- 
nucleus.  Schaudinn  calls  it  the  locomotor  nucleus,  and  says  it  produces 
undulatory  membrane  and  flagella.  If  secondary  flagella  are  present 
they  are  generally  directed  backward. 

The  undulating  membrane  extends  along  one  side  of  the  organism 
from  the  "flagellum  root"  or  near  it  to  the  anterior  end  of  the  parasite, 
whence  it  continues  as  the  free  flagellum.  It  varies  in  size  and  fulness 
according  to  age  and  species. 

The  nucleus  is  situated  anteriorly;  it  is  granular,  thick,  and  egg- 
shaped,  but  varies  somewhat  in  size  and  shape.  The  cytoplasm  is  homo- 
geneous or  granular,  varying  with  age,  environment,  and  possibly 
species.  The  life  cycle  is  not  well  known.  Multiplication  occurs  through 
longitudinal  division;  however,  MacNeal  states  that  the  division  is  not 
exactly  longitudinal,  but  always  more  or  less  oblique  in  direction,  and 
that  the  flagellum  does  not  divide,  but  a  new  one  is  formed  in  each 
division.  Conjugation  has  been  observed  in  trypanosoma  Brucei  by 
Plimmer  and  Bradford. 

Only  a  few  of  the  many  species  of  trypanosomes  described  are  patho- 
genic, and  these  principally  for  the  lower  animals,  though  recently  the 
organisms  have  several  times  been  seen  in  human  beings,  accompanied 
or  not  by  pathological  changes.  Very  recently  Castellani  stated  that 
the  sleeping  sickness  of  the  negro  is  caused  by  a  trypanosome.  The 
work  of  Schaudinn  and  of  Novy  and  MacNeal  make  it  evident  that  the 
spirochsetes  of  recurrent  fever  and  of  geese  belong  to  the  trypanosomes. 

The  first  species  of  trypanosomes  studied  with  any  degree  of  fulness  is 
the  comparatively  non-pathogenic  trypanosoma  Lewisi,  Kent.  It  is  of 
interest  because  of  its  similarity  to  the  more  pathogenic  forms  and  because 
of  the  ease  with  which  it  may  be  studied.  It  is  parasitic  in  the  blood  of 

1  Special  literature  on  trypanosoma :    Laveran  and   Mesnil,  Musgrave  and   Clegg,  Novy  and 
MacNeal,  and  Schaudinn. 


TRYPANOSOMA 


483 


about  25  per  cent,  of  wild  rats  in  almost  all  parts  of  the  world.    It  is  found 
less  frequently  in  tame  rats,  especially  in  the  white  variety.   It  occasionally 


FIG.  146 


Trypanosoma  Lewisi  (Kent).    From  the  blood  of  a  rat.    (Kempner  and  Rabinowitsch.) 

FIG.  147 


Agglutination  of  Trypanosoma  Lewisi.    (Laveran  and  MesniL) 

produces  sickness  and  death  and  even  small  epidemics,  but  generally 
it  is  found  in  apparently  healthy  animals.  A  morphologically  similar 
trypanosome  is  found  in  hamsters,  but  as  neither  variety  will  ^ro\v  in 


484  PROTOZOA 

the  blood  of  the  other  host  they  must  be  regarded  as  physiological 
varieties. 

These  flagellatawere  probably  first  seen  in  the  blood  of  the  rat  in  1845, 
but  they  were  not  well  described  until  1879,  when  Lewis  studied  them 
more  fully.  Since  then  they  have  been  studied  by  many  observers, 
especially  by  Kempner  and  Rabinowitsch,  Wasielewski  and  Senn, 
Jiirgens,  Laveran  and  Mesnil,  and  Novy  and  MacNeal. 

Morphology. — Their  length,  including  the  flagellum,  is  from  8[t  to  30/*, 
their  breadth  2p  to  3/Jt.  The  body  is  lance-shaped  and  shows  a  pro- 
toplasm finely  granular  in  the  young  form,  more  coarsely  so  as  age 
increases.  The  single  flagellum  is  almost  as  long  as  the  body  and  arises 
from  the  posterior  third  of  the  organism  in  or  near  a  small,  more  or  less 
oval  body,  the  flagellum  root  (centrosome,  micronucleus  blepharoblast), 
which  during  division  often  divides  first.  The  flagellum  continues  for- 
ward as  the  thickened  edge  of  the  undulating  membrane,  becoming 
free  at  the  anterior  end  of  the  body.  The  large,  oval,  densely  reticular, 
nucleus  lies  generally  in  the  anterior  third  of  the  body.  No  contractile 
vacuoles  have  been  observed. 

Biology. — The  parasite  is  very  motile,  probably  more  so  than  any  other 
variety.  Its  motility  soon  ceases  outside  of  the  body,  continuing  longer  in 
the  ice-box  than  at  higher  temperatures.  Also  unless  kept  at  low  tem- 
peratures the  organism  dies  very  quickly.  It  is  rather  diagnostic  of  it  that 
at  ice-box  temperature  it  lives  longer  than  any  other  variety  of  trypano- 
some  studied.  Long  after  the  organisms  have  lost  their  motility  and 
ability  to  stain  well,  and  even  after  they  seem  to  have  broken  up  com- 
pletely, the  blood  containing  them  is  still  infectious  for  rats.  In  this 
connection  it  is  interesting  to  note  that  the  blood  of  infected  animals  in 
which  no  trypanosoma  can  be  demonstrated  is  infectious  for  fresh  animals. 

Kempner  and  Rabinowitsch  have  succeeded  in  producing  active 
and  passive  immunity.  The  blood  of  immunized  animals  causes  agglu- 
tination of  the  trypanosomes  without  immobilization.  According  to 
Laveran  and  Mesnil  the  serum  possesses  no  lytic  properties  for  th& 
trypanosomes,  and  they  state  that  the  inoculation  of  such  seTtim  intfa^ 
peritoneally  with  active  trypanosomes  seems  simply  to  cause  an  increased 
power  of  the  phagocytes  over  them,  whereas  MacNeal  states  that  the 
serum  does  possess  cytolytic  properties  for  the  parasites. 

Recently,  Novy  and  MacNeal  have  reported  the  artificial  cultivation 
of  the  rat  trypanosome.  At  room  temperature  they  have  grown  the 
organism  through  eleven  culture  generations  in  test-tubes  for  an  entire 
year.  At  the  end  of  this  time  the  parasites  were  as  virulent  as  at  the 
beginning.  The  culture  medium  used  in  their  work  was  ordinary 
nutrient  agar  containing  variable  amounts  of  fresh  defibrinated  or  laked 
rabbit  or  rat  blood.  The  best  results  were  obtained  with  a  mixture  of 
two  parts  of  the  blood  to  one  of  agar,  though  growth  was  obtained  on 
dilutions  as  high  as  one  part  of  blood  to  ten  of  agar.  At  room  tem- 
perature the  growth  is  slower  but  surer  than  in  the  thermostat.  The 
cultures  at  room  temperature  retain  their  vitality  for  months;  thus  in 
one  case  the  trypanosomes  were  alive  after  three  hundred  and  six  days. 


TRYPANOSOMA  435 

These  results  have  been  corroborated  by  Kempner  and  Rabinowitsch, 
Laveran  and  Mesnil,  and  by  ourselves. 

Trypanosoma  Evansi  (Steel). 

The  next  trypanosome  of  importance  studied,  and  the  first  of  the 
more  pathogenic  trypanosomes,  is  trypanosoma  Evansi,  Steel.  This 
species  was  discovered  by  Evans  in  1880  in  the  blood  of  horses  suf- 
fering with  the  disease  known  as  surra,  in  India.  Lingard's  important 
work  on  this  disease  in  1893  led,  in  a  way,  to  all  the  subsequent  work 
on  diseases  caused  by  trypanosomes.  In  general  the  descriptions  given 
of  the  symptomatology  of  trypanosomiasis  in  various  animals  show 
a  great  similarity,  though  there  is  much  variation  in  individual  cases. 
In  a  well-established  infection  the  clinical  picture-  according  to  Mus- 
grave  and  Clegg  is  as  follows :  After  an  incubation  period  which  varies 
in  the  same  class  of  animals  and  in  those  of  different  species,  as  well  as 
with  the  conditions  of  infection,  and  during  which  the  animal  remains 
perfectly  well,  the  first  symptom  to  be  noticed  is  a  rise  of  temperature; 
and  for  some  days  a  remittent  or  intermittent  fever  may  be  the  only 
evidence  of  illness.  Later  on  the  animal  becomes  somewhat  stupid; 
watery,  catarrhal  discharges  from  the  nose  and  eyes  appear;  the  hair 
becomes  roughened  and  falls  out  in  places.  Finally  the  catarrhal  dis- 
charges become  more  profuse  and  the  secretions  more  tenacious  and 
even  purulent;  marked  emaciation  develops;  oedema  of  the  genitals 
and  dependent  parts  appears ;  a  staggering  gait,  particularly  of  the  hind 
parts,  comes  on,  and  is  followed  by  death.  There  may  be  various 
ecchymoses  and  skin  eruptions.  Parasites  are  found  in  the  blood  more 
or  less  regularly  after  the  appearance  of  the  fever. 

The  autopsy  generally  shows  anaemia,  an  enlarged  spleen  with  hyper- 
trophied  follicles,  more  or  less  gelatinous  material  in  the  adipose  tissue, 
the  liver  slightly  enlarged,  a  small  amount  of  serous  exudate  in  serous 
cavities,  oedematous  condition,  and  small  hemorrhages  in  various  tissues. 

The  duration  varies  from  a  few  days  to  many  months.  The  prognosis 
seems  to  be  influenced  to  a  certain  extent  by  the  species  of  host.  It  is 
probably  always  fatal  in  horses.  Some  cattle  recover.  The  cause  of 
death  is  possibly  a  toxic  substance,  though  no  definite  toxin  has  been 
isolated.  Mechanical  disturbances  (emboli,  etc.)  also  probably  play  a 
part  in  producing  death.  The  hosts  of  trypanosoma  Evansi  are  horses, 
mules,  cattle,  camels,  elephants,  buffaloes,  and,  according  to  Musgrave 
and  Clegg,  rats.  After  experimental  inoculation  this  trypanosome  is 
infectious  for  dogs,  monkeys,  rabbits,  guinea-pigs,  mice,  and  cats. 
Man  seems  to  be  immune.  It  is  without  doubt  transmitted  from  animal 
to  animal  by  the  bites  of  insects  (flies  and  fleas). 

Besides  the  differences  in  virulence,  the  trypanosoma  Evansi  is  dif- 
ferentiated morphologically  from  the  trypanosoma  Lewisi  by  a  larger 
average  length  (20//  to30/^  long  and  !/>«  to  2//  wide).  It  differs  from 
the  trypanosoma  Brucei  in  having  a  more  pointed  posterior  end.  Many 
authors,  however,  consider  it  identical  with  the  latter  species. 


486  PROTOZOA 

Trypanosoma  Brucei  (Plimmer  and  Bradford) . 

The  trypanosoma  Brucei  (Plimmer  and  Bradford)  was  discovered 
by  Bruce  in  1894  in  the  blood  of  horses  and  cattle  suffering  from  nagana 
in  Zululand  and  other  parts  of  Africa.  Bruce  demonstrated  that  the 
contagion  was  caused  by  the  bites  of  a  fly,  the  Glossina  morsitans,  or 
tsetse-fly.  Since  then  other  varieties  of  flies  also  have  been  shown  to 
spread  the  disease.  These  flies  bite  by  day  and  in  full  moonlight.  The 
infectivity  of  the  insects  lasts  for  about  forty-eight  hours  after  they  have 
bitten  a  sick  animal.  Bruce  found  living  trypanosomes  in  the  proboscedes 
of  the  flies  at  the  end  of  that  time.  Up  to  one  hundred  and  eighteen  hours 
they  were  found  in  the  flies'  stomachs,  but  after  one  hundred  and  forty 
hours  the  stomachs  were  empty  and  what  appeared  to  be  dead  para- 
sites were  found  in  the  excreta.  No  development  in  the  fly  has  been 
observed.  The  disease  is  chronic  enough  in  some  animals  to  account 
for  a  continuous  source  of  infection.  The  natural  hosts  of  this  species 
are  horses,  cattle,  camels,  antelopes,  swine,  and  various  wild  animals. 
According  to  Laveran  and  Mesnil  all  mammifera  are  susceptible  to 
trypanosoma  Brucei,  though  sheep  and  African  goats  seem  to  be 
partial  exceptions.  Men  and  birds  seem  to  be  immune.  Horses  and 
dogs  are  especially  susceptible.  The  incubation  time  in  natural  infec- 
tion is  not  more  than  nine  days.  The  course  and  duration  are  irregular, 
as  in  other  trypanosomatic  diseases. 

Novy  and  MacNeal  have  been  successful  also  in  cultivating  the  try- 
panosoma Brucei  in  vitro,  though  it  is  much  more  exacting  in  its  re- 
quirements than  is  the  trypanosoma  Lewisi.  The  same  methods  are 
used,  but  the  blood  dilution  must  not  be  less  than  two  parts  to  one  of 
nutrient  agar.  These  investigators  state  that  the  cultural  characteristics 
are  such  as  to  enable  perfect  differentiation  between  the  two  trypano- 
somes. For  in  their  cultures  the  trypanosoma  Brucei  have  characteristic 
granules,  the  trypanosoma  Lewisi  have  none;  the  trypanosoma  Brucei 
show  little  variation  in  size  (15;*  to  17/*  in  length),  the  trypanosoma 
Lewisi  vary  so  much  (I/*  to  60ju  long)  that  there  are  forms  small  enough 
to  pass  a  Berkefekl  filter;  the  trypanosoma  Brucei  has  a  slow,  wriggling 
motion,  the  trypanosoma  Lewisi  moves  with  great  rapidity  and  in  an 
almost  straight  line;  and  finally  the  trypanosoma  Brucei  form  small, 
irregular  colonies,  while  the  trypanosoma  Lewisi  form  large  symmetri- 
cal ones. 

The  question  as  to  the  identity  of  the  trypanosoma  Brucei  with  other 
of  the  more  pathogenic  trypanosomes  has  not  yet  been  decided. 

So  far  it^has  not  been  possible  to  immunize  the  more  susceptible 
animals  against  this  species  of  trypanosome.  Sheep,  goats,  and  cattle 
are  less  susceptible  and  in  their  case  recovery  from  the  disease  protects 
against  subsequent  inoculation. 

Novy  and  MacNeal  state  that  older  cultures  of  trypanosoma 
Brucei,  especially  those  exposed  to  a  temperature  of  34°  C.,  become 
less  virulent  and  eventually,  though  living,  fail  to  infect  animals,  and 
they  think  that  repeated  injections  of  these  attenuated  cultures  may 


TRYPANOSOMA  487 

impart  immunity,  and  that  in  this  way  it  may  be  possible  to  secure  pro- 
tection against  the  ravages  of  nagana. 

In  1896  Rouget  discovered  a  trypanosome  in  the  blood  of  bleeding 
equines  in  Algiers  and  South  Africa  affected  by  the  disease  called 
Dourine.  Some  authors  think  this  disease  is  identical  with  nagana 
and  surra,  but  the  fact  that  it  has  been  impossible  to  infect  cattle  with 
the  blood  of  the  sick  equine  point  to  its  being  a  distinct  disease.  The 
trypanosoma  is  called  trypanosoma  equiperdum  or  trypanosoma  Rougeti. 

Recently,  mal  de  caderas,  a  disease  of  horses,  asses,  and  mules  in 
South  Africa,  having  the  general  characteristics  of  trypanosomiasis, 
has  been  shown  by  Voges  to  be  due  to  trypanosornes.  He  called 
this  organism  trypanosoma  equinum,  and  believes  it  to  be  a  dis- 
tinct species,  resembling  more  closely  the  trypanosoma  Lewisi.  He 
considers  cattle  immune. 

In  1902  Laveran  described  a  variety  of  trypanosome  found  by  Theiler 
to  produce  disease  in  ruminants  of  the  Transvaal.  According  to 
Laveran  and  Mesnil  it  is  characterized  by  having  the  "flagellum  root" 
near  the  centre  of  the  parasite,  near  and  sometimes  united  to  the  nucleus. 
This  variety  has  been  named  trypanosoma  Transvaaliense,  while 
another  variety  found  in  cattle  of  South  Africa  by  Theiler,  and  pro- 
nounced by  Laveran  and  Mesnil  to  be  a  distinct  species,  was  named 
trypanosoma  Theileri. 

Trypanosomes  have  been  found  in  the  blood  of  apparently  normal 
frogs,  fishes,  birds,  guinea-pigs,  rabbits,  and  bats.  No  relationship  has 
been  shown  to  exist  between  these  non-pathogenic  forms  and  those 
causing  disease  in  the  higher  animals. 

Trypanosomes  in  Man. 

In  1898  Nepvieu  reported  having  found  trypanosomes  in  the  blood  of 
6  out  of  more  than  200  cases  examined  for  malarial  organisms.  In  all 
of  these  cases  malarial  organisms  too  were  found,  and  no  symptoms  char- 
acteristic of  the  invasion  of  trypanosomes  were  observed.  Nepvieu  found 
flagellates  in  a  seventh  case  which  was  apparently  in  good  health. 

The  eighth  case  is  reported  by  Dutton  in  1901.  This  case  was  a 
European  who  had  lived  some  years  in  AVest  Africa.  The  principal 
symptoms  were  gradual  wasting  and  weakness;  irregular  temperature, 
never  very  high  and  of  a  relapsing  type ;  local  oedemas,  congested  areas 
of  the  skin,  enlargement  of  the  spleen,  and  constant  increased  frequency 
of  pulse  and  respiration.  It  ended  fatally  after  one  year  and  eight 
months.  The  chronic  character  was  repeated  in  animals.  Some  white 
rats  were  refractory;  others  died  in  two  to  three  months.  In  monkeys 
(Macacus  rhesus)  it  was  fatal  in  about  two  months.  Dogs  were  un- 
affected. This  trypanosome  is  distinctly  smaller  than  the  other  species 
described,  and  there  is  little  doubt  of  it  beinir  a  distinct  species.  Dutton 
also  found  trypanosomes  in  the  blood  of  1  out  of  150  apparently  healthy 
Gambian  children  examined  by  him. 

The  tenth  case  is  published  by  Manson  in  1902.    This  was  a  mis- 


488  PROTOZOA 

sionary's  wife  who  had  resided  on  the  Upper  Congo  for  a  year.  She 
presented  the  same  group  of  symptoms  as  Button's  case,  and  after 
repeated  examinations  trypanosomes  were  found  in  her  blood.  Manson 
soon  after  published  a  similar  case.  Broeden  has  published  2  more 
cases  and  recently  Baker  has  reported  3  cases  among  human  beings. 

Of  these  16  cases  of  trypanosomiasis  in  man,  2  were  apparently 
healthy  persons,  6  had  malarial  fever  as  well,  and  8  were  such  as 
showed  clinical  symptoms  apparently  entirely  due  to  the  infection  with 
trypanosomes. 

Quite  recently  Castellani  has  stated  that  the  cause  of  sleeping  sick- 
ness of  the  negro  is  a  trypanosome.  He  found  trypanosomes  in  the 
centrifugalized  cerebrospinal  fluid  of  20  out  of  34  cases  of  this  disease. 
His  work  has  been  corroborated  by  Bruce,  Nabarro,  Greig  and  others. 
Bruce  found  trypanosomes  in  the  fluid  obtained  by  lumbar  puncture  in 
all  of  the  38  cases  examined  and  in  12  out  of  13  cases  in  the  blood.  The 
trypanosomes  found  in  these  cases  resemble  those  found  in  other  human 
beings,  and  probably  belong  to  the  same  species.  Laveran  and  Mesnil 
recommend  the  names  trypanosoma  Gambiense  Button  for  the  para- 
site and  human  trypanosomiasis  for  the  disease. 

Sleeping  sickness,  or  human  trypanosomiasis,  is  a  disease  of  the  negro, 
endemic  in  certain  regions  of  equatorial  Africa.  Neither  age  nor  sex 
are  predisposing  factors,  but  occupation  and  social  position  seem  to 
have  a  marked  influence,  the  great  majority  of  the  cases  occurring 
among  very  poor  field  workers.  As  these  workers  are  all  negroes,  the 
question  of  the  influence  of  race  cannot  be  determined.  The  white 
race,  however,  is  not  immune,  as  has  been  shown  by  the  cases  quoted 
above. 

In  places  where  most  of  the  cases  occur,  a  fly  belonging  to  the  species 
glossina  (Glossina  palpatis)  is  very  abundant ;  in  places  where  this  fly 
is  not  found  no  cases  occur.  Hence,  it  is  highly  probable  that,  as  'in 
the  trypanosomiasis  of  the  lower  animals,  the  contagion  is  spread  by  a 
biting  insect. 

Symptoms. — The  course  of  the  disease  is  very  insidious,  as  the  try- 
panosomes may  exist  in  the  blood  for  a  long  time  before  entering  and 
growing  in  the  cerebrospinal  fluid  and  causing  the  characteristic  symp- 
toms of  sleeping  sickness.  Therefore,  the  symptoms  may  be  divided 
into  two  stages.  In  the  first  stage  there  is  only  an  irregular  fever.  In 
the  second  stage  the  fever  becomes  hectic,  the  pulse  is  constantly  in- 
creased; there  are  neuralgic  pains,  partial  cedemas  and  erythemas, 
trembling  of  the  muscles,  gradually  increasing  weakness,  emaciation, 
and  lethargy.  The  somnolence  increases  until  a  comatose  condition  is 
developed  and  death  occurs.  In  the  second  stage  trypanosomes  are 
always  found  in  the  cerebrospinal  fluid.  Throughout  the  disease  they 
are  usually  found  in  small  numbers  in  the  blood. 

Duration. — The  first  stage  may  last  for  several  years;  the  second, 
from  four  to  eight  months.  The  percentage  of  deaths  in  cases  reaching 
the  second  stage  is  100.  Whether  some  in  the  first  stage  recover  is  not 
yet  certain. 


TRYPANOSOMA  489 

The  trypanosoma  Gambiense  is  irregularly  pathogenic  for  some 
monkeys  (Macacus  rhesus  and  others),  for  dogs,  cats,  and  rats.  It  is 
less  pathogenic  for  mice,  guinea-pigs,  rabbits,  and  horses.  Cattle  and 
swine  seem  to  be  refractory. 

Pathological  Anatomy. — Congestion  of  the  meninges;  increased  quan- 
tity of  cerebrospinal  fluid ;  hypertrophy  of  spleen,  liver,  and  lymphatic 
ganglia. 

Methods  of  Examination.  BLOOD. — If  the  direct  examination  of  the 
blood  is  negative,  10  c.c.  should  be  withdrawn  from  a  yein,  and  after 
a<  Id  ing  a  tenth  of  its  volume  of  citrate  of  sodium  it  should  be  centrifuged 
for  ten  minutes,  and  the  sediment  examined  in  hanging  drop  and  in  smear. 

CEREBROSPINAL  FLUID. — Ten  c.c.  of  the  fluid  withdrawn  by  lumbar 
puncture  should  be  centrifuged  for  fifteen  minutes  and  the  deposit 
should  be  examined  under  150  to  200  diameter  magnification.  Inocu- 
lation of  susceptible  animals  should  also  be  made  with  the  blood  or 
cerebrospinal  fluid  from  the  suspected  case. 

So  far  Musgrave  and  Clegg,  after  many  careful  examinations,  have 
not  found  trypanosoma  in  any  human  beings  in  the  Philippines. 

Diagnosis  of  Trypanosomiasis  in  General. — This  should  be  made  as 
early,  as  possible  in  order  to  prevent  the  spread  of  the  disease.  An 
early  positive  diagnosis  can  only  be  made  by  the  determination  of  the 
blood  infection.  This  is  done  in  two  ways:  first,  by  the  microscopic 
examination  of  a  hanging  drop  of  freshly  drawn  blood;  second,  by 
animal  inoculation.  In  the  microscopic  examination  it  may  be  neces- 
sary to  examine  the  blood  of  the  suspected  animal  for  several  days  in 
succession.  The  parasites  are  rarely  absent  in  the  early  stages  in  domes- 
tic animals  for  more  than  a  few  days  at  a  time,  while  in  man  the  time 
may  be  much  longer.  If  the  trypanosomes  cannot  be  found  by  this 
method,  animal  experiment  should  always  be  made.  Monkeys,  if 
possible,  should  be  used,  or  if  monkeys  cannot  be  obtained,  dogs  or  rats 
may  be  used.  A  few  drops  to  1  c.c.  of  the  blood  from  the  suspected 
animal  should  be  inoculated  intraperitoneally  or  subcutaneously. 

Blood  smears  may  be  stained  by  any  modification  of  the  Romanowsky 
method. 

Prophylaxis  against  Animal  Trypanosomiasis. — The  disease  is  readily 
controlled  by  preventive  measures.  There  should  be  strict  quarantine 
regulations  governing  the  importation  of  animals.  When  the  disease  has 
once  appeared  the  following  general  measures  should  be  taken:  1.  Sus- 
pected animals  should  be  isolated.  2.  All  infected  animals  should  be 
destroyed.  3.  As  far  as  possible  all  biting  insects  should  be  destroyed. 
4.  The  bodies  of  infected  animals  should  be  protected  from  biting 
insects  for  at  least  twenty-four  hours  after  death.  5.  Susceptible  ani- 
mals should  if  possible  be  made  immune. 

Treatment. — Many  drugs  have  been  tried  without  success.  Arsenic 
in  various  forms  has  been  found  to  prolong  life,  but  has  produced  no 
cures.  It  is,  therefore,  not  to  be  recommended  for  the  lower  animals. 
Recently,  Ehrlich  and  Shiga  have  found  that  a  certain  red  product  of 
the  benzopurpurine  series,  to  which  they  have  given  the  name  "  trypan- 


490  PROTOZOA 

roth/'  has  a  preventive  and  curative  effect  in  mice  infected  with  mal  de 
coder  as.  The  curative  effect  is  especially  marked.  As  late  as  three  days 
after  infection  with  the  trypanosome  cures  are  effected.  Inasmuch  as 
the  "  trypanroth"  is  non-poisonous  for  the  trypanosome  in  vitro,  Ehrlich 
and  Shiga  suppose  that  a  toxic  substance  is  formed  in  the  mice.  The 
preventive  effect  of  the  trypanroth  soon  passes  off,  allowing  the  mice  to 
become  infected  with  trypanosomes  two  to  three  days  after  a  preventive 
inoculation.  On  other  trypanosomes  and  in  other  animals  the  results 
are  not  so  good.  Alternating  arsenic  and  "  trypanroth "  may  give 
better  results. 

Serum  Therapy. — Various  normal  sera  from  different  animals  have  been 
tried,  with  practically  no  success.  A  few  have  prolonged  life.  Thus 
Laveran  and  Mesnil  state  that  human  serum  injected  in  sufficient 
quantities  shows  manifest  action  on  the  disease,  and  that  sometimes 
cure  results  in  mice  and  rats.  Further,  by  alternating  human  serum 
with  arsenic  they  obtained  better  results  still.  Kanthack,  Durham,  and 
Blandford  showed  that  animals  recovering  from  trypanosoma  infection 
were  immune  to  further  infection.  Rabinowitsch  and  Kempner  have 
made  a  very  careful  study  of  immune  serum  produced  by  the  trypano- 
soma Lewisi.  Not  only  have  they  shown  that  an  animal  may  be  hyper- 
immunized  and  that  then  its  serum  in  comparatively  large  doses  inocu- 
lated into  mice  at  the  same  time  as  the  trypanosomes,  or  twenty-four 
hours  before  or  after,  allows  no  development  of  the  organisms;  but 
also  Laveran  and  Mesnil  state  that  the  serum  causes  their  rapid 
destruction  by  the  leukocytes,  though  MacNeal,  on  the  other  hand, 
states  that  they  are  destroyed  by  a  cytolytic  action  of  the  serum.  This 
immune  serum  also  has  a  similar  action  on  the  trypanosoma  of  Dourine. 
The  serum  of  animals  hyperimmunized  against  other  varieties  of  try- 
panosoma is  not  as  active  as  that  obtained  by  the  inoculation  of 
trypanosoma  Lewisi,  mal  de  caderas  giving  the  best  results  so  far,  but 
results  that  are  not  encouraging  for  practical  treatment. 

Koch  suggested  that  an  immunity  might  be  established  by  the  inocu- 
lation of  attenuated  parasites,  and  Novy  and  MacNeal  have  succeeded 
in  attenuating  cultures  of  trypanosoma  Brucei,  and  have  obtained  some 
success  in  protecting  experimental  animals  against  virulent  cutures. 

Spirochsete  Obermeieri  (Spirillum  of  Relapsing  Fever). 

Until  very  recently  this  organism  was  classed  with  the  bacteria,  but 
it  is  now  placed  by  Schaudinn  and  others  with  the  flagellates,  as  it  has 
many  of  the  characteristics  of  the  trypanosomes. 

This  spirochaete  was  first  observed  by  Obermeier  in  1873  in  the 
blood  of  persons  suffering  from  relapsing  fever.  It  was  found  in  large 
numbers  during  the  height  of  the  fever,  it  disappeared  about  the  time 
of  the  crisis,  and  reappeared  during  the  relapses.  It  was  not  found  in 
other  diseases.  Obermeier  considered  it  the  cause,  of  the  disease,  and 
his  views  were  shown  to  be  correct  by  the  production  of  the  disease  in 
man  and  ape  through  experimental  inoculation. 


TRYPANOSOMA 


491 


Flo.  148 


Morphology.— The  organisms  are  long,  slender,  flexible,  spiral  or 
wavy  filaments  with  pointed  ends,  from  16/^  to  40/^  in  length  and 
from  one-quarter  to  one-third  the  thickness  of  the  cholera  spirillum 
(Fig.  148).  They  stain  somewhat  faintly  with  watery  solutions  of  the 
basic  aniline  dyes,  better  with  Loeffler's  or  Kiihne's  methylene-blue 
solutions,  or  with  carbol  fuchsin;  best  with  the  Romanowsky  method 
or  its  modifications.  They  do  not  stain  by  Gram's  method. 

Biological  Characters.— In  fresh  preparations  from  the  blood  the 
spirocheetes  exhibit  active  progressive  movements  accompanied  by 
very  rapid  rotation  in  the  long  axis  of  the  spiral  filaments  or  by  undu- 
lating movements.  They  are  found  only  in  the  blood  or  blood' organs, 
never  in  the  secretions,  and  only  during  the  fever,  not  in  the  intermis- 
sions, or  at  most  singly  at  the  begin- 
ning of  or  for  a  short  time  after  an 
attack. 

When  kept  in  blood  serum  or  a  0.6 
per  cent,  solution  of  sodium  chloride 
they  continue  to  exhibit  active  move- 
ments for  a  considerable  time.  They 
may  be  preserved  alive  and  active  for 
many  days  in  sealed  tubes.  They  are 
killed  quickly  at  60°  C.,  but  they  re- 
main alive  for  some  time  at  0°  C. 
Efforts  to  cultivate  them  in  artificial 
culture  media  have  thus  far  been 
unsuccessful,  although  Koch  has  ob- 
served an  increase  in  the  length  of  the 
spirilla  and  the  formation  of  a  tangled  and 
mass  of  filaments.  But  now  with  the 
cultivation  of  trypanosomes  in  vitro  by  Novy  and  MacNeal  successful, 
one  may  hope  for  similar  results  with  the  spirochsete  Obermeieri. 

Pathogenesis. — In  man,  whether  the  disease  is  acquired  naturally 
or  by  artificial  inoculation,  the  organism  causes  the  following  symp- 
toms :  After  a  short  period  of  incubation  the  temperature  rises  rapidly, 
remains  high  for  five  to  seven  days,  and  then  returns  to  normal  by 
crisis.  About  seven  days  later  there  is  another  sudden  rise  of  tem- 
perature, but  this  time  the  crisis  occurs  sooner.  A  second  or  third 
relapse  may  occur.  The  organisms  increase  in  numbers  rapidly  in 
the  blood  from  the  beginning  of  the  fever,  large  numbers  often  being 
found  in  every  microscopic  field.  They  begin  to  disappear  a  short 
time  before  the  crisis,  and  immediately  after  the  crisis  it  is  practically 
impossible  to  find  them  in  the  circulating  blood.  The  mortality  varies 
in  different  epidemics  from  2  to  10  per  cent.  When  monkeys  are 
inoculated  with  human  blood  containing  the  spirilla  they  become 
sick  about  three  and  a  half  days  later,  but  show  only  the  initial  febrile 
attack,  or,  at  the  most,  an  occasional  short  relapse.  The  organisms 
are  found  to  have  the  same  relation  to  the  pyrexial  period  as  in  man. 
Blood  from  one  animal  taken  during  the  fever  induces  a  similar  febrile 
paroxysm  when  inoculated  into  another  animal. 


Spirochsete    Obermeieri    blood    smear. 
Fuchsin.     x  1000  diam.     (From  Itzerott 


492  PROTOZOA 

Metchnikoff  showed  that  during  the  intermissions  when  the  spirilla 
disappeared  from  the  circulating  blood  they  accumulated  in  the  spleen 
and  were  ingested  in  large  numbers  by  certain  phagocytes  and  finally 
were  destroyed. 

According  to  Lamb  a  certain  amount  of  immunity  is  conferred  upon 
monkeys  (Macacus  radiatus)  soon  after  an  attack,  but  it  disappears 
quickly.  If  the  serum  is  removed  during  this  time  it  is  found  to  have 
some  protective  action  when  mixed  with  the  blood  containing  spirillse, 
and  also  to  cause  agglutination  of  the  organisms. 

Infection  probably  occurs  through  the  bite  of  blood-sucking  insects. 

Button  showed  (1905)  that  tick  fever  of  the  Congo,  which  is  caused 
by  an  organism  similar  to  that  causing  relapsing  fever  elsewhere,  can 
be  transferred  to  monkeys  by  the  bites  of  young  ticks  at  their  first  feed 
after  hatching  from  infected  parents.  He  accidentally  demonstrated 
the  fact  that  the  disease  can  be  inoculated  into  human  beings  through 
a  cut  surface,  for  after  a  wound  received  at  autopsy  he  developed  the 
disease  which  eventually  caused  his  death. 

Spirilla  similar  to  the  spirochsete  Obermeieri  have  been  found  in 
birds. 

Spirochaete  Pallida  Schaudinn  in  Syphilis. 

The  first  two  papers  by  Schaudinn  and  Hoffmann1  appeared  almost 
simultaneously.  The  paper  in  the  former  publication  was  illustrated 
with  two  photomicrographs,  showing  the  form  of  the  organism.  These 
papers  were  quickly  followed  by  two  communications  from  Metchni- 
koff's2  laboratory  in  the  Pasteur  Institute,  confirming  and  accepting 
the  discovery,  and  drawing  attention  to  the  interesting  fact  that  Bordet 
and  Gengou  had  observed  the  same  micro-organism  in  a  syphilitic 
chancre  some  three  years  before.  However,  as  they  failed  to  discover 
it  in  some  syphilitic  lesions  which  they  subsequently  studied  they 
abandoned  any  future  search  for  it.  In  this  country  the  results  of  the 
above  investigators  have  been  corroborated  by  Flexner  and  Ewing. 

The  first  publication  of  Hoffmann  and  Schaudinn  dealt  with  a  study 
of  primary  chancres,  the  enlarged  glands  of  the  groin  attending  these 
lesions,  and  flat  condylomata  in  syphilitic  patients.  The  study  con- 
sisted in  the  examination  of  fresh  specimens  obtained  from  the  surface 
and  interior  of  the  primary  lesions  and  the  interior  of  lymph  glands 
and  condylomata,  and  stained  specimens  from  the  same  sources.  Certain 
control  examinations  were  also  made  of  non-syphilitic  lesions  of  the 
genitals  and  of  mixed  lesions  of  these  parts.  The  results  were  quite 
uniform  and  suggestive.  From  the  cases  of  simple  syphilitic  infection 
the  lymph  glands,  condylomata,  and  interior  of  chancres  showed  a 
variable  number  of  spiral  micro-organisms  of  great  tenuity,  for  which 
they  propose  tentatively  the  name  spirochrete  pallida,  while  the  non- 

1  Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte,  Berlin,  April  10, 1905,  xxii.;  Zweite's  Heft,  527  ; 
Deutsche  medizinische  Wochenschrift,  May  4, 1905,  xxxi.  p.  711. 

2  Metchnikoff  and  Roux,  Recherches  microbiologiques  sur  sa  syphilis,  Bulletin  de  1' Academic  de 
medecine,  Paris,  May  16, 1905. 


TRYPANOSOMA 


493 


FIG.  149 


specific  lesions  showed  a  second  spiral  micro-organism,  for  which  they 
propose  the  name  of  spirochaete  refringens  (Fig.  149)  .  The  latter  organ- 
ism had,  doubtless,  been  seen  and  described  before  by  several  observers. 
Schaudinn  and  Hoffmann  did  not  find  the  first  spirochsete  in  non- 
syphilitic  lesions,  nor  did  they  find  the  second  in  the  interior  of  the 
syphilitic  lesions  studied  by  them.  From  the  study  of  Schaudinn  and 
Hoffmann  it  is  not  difficult  to  explain  the  failure  of  previous  investigators 
to  perceive  spirochaete  pallida  and  especially  the  failure  of  Bordet  and 
Gengou  to  obtain  it  in  all  of  the  several  cases  studied  by  them.  The 
organism  is  difficult  to  see  in  the  fresh  state,  and  it  is  also  highly  refrac- 
tory to  staining,  so  that  special  methods  are  required  to  demonstrate 
it  in  fixed  preparations.  The  description  of  the  organism  is  as  follows: 
In  the  length  the  spirochaete  varies  from  4/t  to  W/JL,  the  average  being 
7ft;  in  width  the  variation  is  from  immeasur- 
able thinness  to  J/A  The  number  of  bends 
is  from  3  to  12.  The  organism  agrees  in 
motility  with  the  spirochaetes  rather  than 
with  the  spirilla;  there  'are  three  character- 
istic movements:  rotation  on  the  long  axis, 
forward  and  backward  motion,  and  bending 
of  the  entire  body.  There  are  indications 
of  an  undulating  membrane,  but  none  of 
flagella.  The  poles  end  in  sharp  points. 
No  further  details  of  structure  have  been 
made  out  thus  far. 

For  the  purpose  of  the  study  of  the  fresh 
material  dilution  with  salt  solution  of  the 
expressed  juices  of  primary  lesions,  or  the 
fluid  drawn  by  aspiration  from  the  lymph 
glands,  is  permissible.  Prepared  in  this  way 
the  spirochaetes  were  still  actively  motile, 
according  to  Schaudinn  and  Hoffmann,  after 

six  hours. 

The    Staining    is    accomplished    with    diffi- 

& 

culty,  and   the  best   results  thus  far  have 

been   obtained   with  Giemsa's    eosin   solution    and    azur. 

and  Hoffmann  recommend  the  following  formula: 

Twelve  parts  of  Giemsa's  eosin  solution  (2.5  c.c.  1  per  cent,  eosin, 
500  c.c.  water). 

Three  parts  azur  No.  I  (1:  1000  solution  in  water). 

Three  parts  azur  No.  II  (0.8:  1000  solution  in  water). 

This  mixture  is  to  be  freshly  prepared.  The  films,  which  should  be 
thinly  spread,  are  dried  in  the  air  and  then  hardened  in  absolute  alcohol 
for  ten  minutes,  after  which  they  are  immersed  in  the  stain  from  six- 
teen to  twenty-four  hours.  They  are  to  be  washed  in  water,  dried  in 
the  air,  and  examined  in  cedar  oil. 

Flexner  and  Noguchi  have  published  a  report  upon  the^examination 
of  four  cases  showing  syphilitic  lesions  an-l  two  controls. 


The  two  spirocba-tes  in  the  centre 
are  Sp.  pallida ;  the  three  others, 
Sp.  refringens.  (Schaudinn  and 
Hoffmann.) 


Schaudinn 


494  PROTOZOA 

Case  I.  Male,  aged  twenty  years.  Luetic  infection  December,  1904. 
No  regular  treatment.  He  presented  mucous  patches  of  the  tonsils 
and  soft  palate,  and  a  fading  rash  of  the  trunk.  Between  the  buttocks, 
flat  condylomata.  A  condyloma  was  excised.  Smears  were  made  and 
stained  in  various  anilines  and  with  eosin-azur.  Fresh  preparations  in 
salt  solution  were  also  studied.  In  the  latter  no  characteristic  organ- 
isms were  found.  The  stained  preparations  were  positive,  showing  a 
variable  number  of  thin,  lightly  stained  spiral  organisms  identified 
as  the  species  described  by  Schaudinn  and  Hoffmann.  The  positive 
results  were  obtained  with  aniline-water-gentian  violet  and  eosin-azur, 
the  latter  having  given  the  most  satisfactory  results.  The  films  varied 
greatly,  even  with  the  same  method  of  preparation.  In  some  the  num- 
ber of  stained  spirals  was  very  small,  while  in  others  the  number  was 
large,  a  single  field  showing  as  many  as  five.  In  still  other  cover- 
glasses  no  organisms  could  be  discovered.  The  spirals  were  long  for 
the  most  part  and  showed  from  six  to  twelve  bends  or  curves. 

Case  II.  Male,  aged  thirty-four  years.  Burrowing  non-syphilitic 
ulcer  of  the  penis.  Smears  from  the  surface  only  could  be  obtained, 
and  they  were  negative  for  spiral  micro-organisms. 

Case  III.  Male,  aged  twenty-three  years.  Mucous  patches  in  mouth. 
Healed  scar  on  penis.  Enlargement  and  induration  of  glands  of  the 
groin.  A  small  quantity  of  fluid,  consisting  of  blood  and  lymph  cells 
aspired  from  the  enlarged  glands.  Fresh  preparations  negative;  smears 
stained  in  eosin-azur  showed  a  very  few  delicate,  faintly-staining  spiral 
organisms. 

Case  IV.  Male,  aged  eighteen  years.  Infection  in  January.  For 
several  weeks  active  antisyphilitic  treatment.  Primary  lesion  on  glans 
penis;  marked  swelling  of  the  glands  of  the  groin.  Part  of  primary 
lesion  was  excised  and  fluid  was  aspirated  from  an  enlarged  gland. 
In  the  fresh  fluid  of  the  gland  diluted  in  sterile  salt  solution  several 
small  and  a  single  larger  motile  spiral  organism  were  seen.  None  of 
the  stained  preparations  from  the  primary  lesion  or  the  gland  juice 
showed  spirochsetes. 

^  Case  V.  Colored  male,  aged  twenty  years.  No  definite  history  of 
time  of  infection.  Presented  himself  with  phimosis  and  balanitis. 
After  incision  of  the  prepuce  three  separate  elevated,  indurated  lesions 
regarded  as  chancres  appeared.  Moderate  swelling  of  the  glands  of  the 
groin.  One  of  the  lesions  excised.  Smears  and  fresh  preparations 
were  made  from  the  base  of  the  lesion.  Fresh  preparations  negative. 
The  smears  stained  in  eosin-azur  showed  a  moderate  number  of  delicate 
spirals,  agreeing  with  the  description  given  by  Schaudinn  and  Hoff- 
mann. They  varied  in  number  in  different  cover-glasses,  and  in  some 
could  not  be  found.  The  curves  numbered  from  eight  to  twelve. 

Case  VI.  Male,  aged  twenty-three  years.  Non-syphilitic  ulcer  of 
penis;  duration  twelve  days.  Films  stained  in  eosin-azur  were  nega- 
tive for  spirochsetes. 

Of  the  four  cases  of  syphilitic  lesions  studied  by  Flexner  and  Noguchi 
the  spiral  organisms  were  obtained  in  stained  preparations  three  times, 


CERCOMOXAS  AND  TRICHOMONAS  495 

while  in  the  two  cases  of  non-syphilitic  lesions  studied  they  could  not  be 
found.  An  anomaly  exists  in  respect  to  Case  IV.,  in  which  the  spirals 
were  missed  in  stained  preparations,  while  they  appeared  to  be  present 
in  the  fresh  state.  No  explanation  can  now  be  offered  for  this  occurrence. 

Schaudinn  and  Hoffmann  express  themselves  very  guardedly  regard- 
ing the  significance  of  the  spirochaete.  They  point  out  its  presence  in 
the  typical  lesions  of  the  disease  and  its  absence  in  the  other  forms  of 
venereal  disease  studied.  Important  confirmatory  contributions  have 
come  from  Metchnikoff  and  Roux,  who  have  demonstrated  the  same 
organism  in  the  lesions  of  acquired  syphilis  in  man  and  in  experimental 
syphilis  in  the  monkey  and  ape.  In  the  last  animals  the  material  for 
study  was  obtained  from  the  primary  lesions  produced  by  inoculation 
before  ulceration  had  taken  place.  Additional  confirmatory  evidence 
of  importance  as  regards  the  distribution  of  the  spirochaete  is  supplied 
by  the  observation  of  Levaditi  and  Buschke  and  Fischer  upon  con- 
genital syphilis.  These  writers  found  that  organism  in  the  pemphigus 
bullae  and  papules  of  the  skin,  and,  in  cases  coming  to  autopsy,  in 
films  from  the  spleen  and  liver.  Schaudinn  reports  that  he  has 
obtained  it  also  from  splenic  juice  removed  by  aspiration  from  a 
syphilitic  patient. 

Metchnikoff  and  Roux  draw  attention  to  the  irregularity  of  distribu- 
tion of  the  organism  as  indicated  by  the  variation  in  numbers  upon  the 
cover-slips.  Others  have  observed  the  same  irregularity,  but  it  is  not 
certainly  established  that  the  difference  may  not  be  due  to  the  imper- 
fect technique  in  staining.  Metchnikoff  and  Roux  and  Levaditi  prefer 
a  more  rapid  method  of  staining  the  films,  namely,  that  of  Marino, 
which,  up  to  the  present,  we  have  used  but  little.  Should  it  serve  as 
good  a  purpose  as  the  slower  eosin-azur,  and  should  future  study  con- 
firm the  etiological  position  of  the  spirochaete,  a  rapid  and  useful  and 
perhaps  even  a  specific  method  of  diagnosis  would  be  afforded.  Since 
the  organism  exists  in  the  primary  lesions  and  the  glands  of  the  groin 
in  a  demonstrable  form,  and  since  fluid  from  each  can  be  obtained 
easily,  with  the  infliction  of  little  pain  to  the  patient,  and  without  in 
any  way  prejudicing  the  progress  of  his  disease,  we  may  look  for  a 
general  study  of  the  fluids  obtained  from  these  sources  in  suspicious 
and  established  forms  of  venereal  disease  with  reference  both  to  the 
occurrence  and  the  specificity  of  spirochaete  pallida. 


CERCOMONAS. 

Subclass:  Flagellata. 

Order:  Protomonadina. 

Genus:  Cercomonas. 

The  members  of  this  genus  are  round  or  oval  flagellates  with  a  long 
anterior  flagellum  and  a  more  or  less  pointed  posterior  one  which  is 
sometimes  amoeboid.  The  vesicular  nucleus  is  situated  anteriorly  and 
lying  near  it  are  one  or  two  contractile  vacuoles.  Division  into  two 
daughter  forms  has  been  observed. 


496 


PROTOZOA 


A  number  of  cercomonada,  none  of  them  well  studied,  have  been 
observed  in  different  animals,  as  well  as  in  man. 

Cercomonas  hominis  (Davaine,  1854)  was  observed  in  the  dejec- 
tions of  a  cholera  patient  by  Davaine.  The  body  is  10/^  to  12/J.  long 
and  pear-shaped,  pointed  posteriorly.  The  flagellum  is  twice  as  long 
as  the  body.  The  nucleus  is  difficult  to  see.  Davaine  also  reported 
a  smaller  form  in  the  stools  of  a  typhoid  patient.  Other  observers  have 
noticed  similar  forms  in  human  stools,  some  associated  with  amoeba 
coli.  Similar  forms  have  been  seen  also  in  an  echinococcus  cyst  of  the 
liver,  in  the  sputum  from  a  case  of  lung  gangrene,  in  the  exudate  of  a 
hydropneumothorax,  and  a  few  times  in  the  urine  (bodo  urinarius). 
They  are  all  probably  harmless  comensals. 

Polymastigina. 

The  order  polymastigina  consists  of  flagellates  having  several  flagella 
projecting  from  different  parts  of  the  body.  The  majority  of  the  forms 
known  are  parasitic  in  certain  fish. 

Donne*  in  1837  described  a  form  belonging  to  this  group  which  he 
found  in  the  human  vagina,  and  which  he  therefore  called  trichomonas 
vaginalis.  It  has  been  found  by  other  observers  to  be  a  frequent  habitant 


FIG. 150 


FIG.  151 


Trichomonas  vaginalis.    (Blochmann.) 


Lamblia  intestinalis.    (Schewiakoff.) 


of  the  vagina  at  all  ages.  It  has  also  been  found  a  few  times  in  the  acid 
urine  of  males.  The  mode  of  infection  of  the  female  is  unknown.  The 
body  of  the  parasite  at  rest  is  pear-shaped,  but  during  action  its  amreboid 
movements  cause  it  to  assume  various  shapes.  The  size  varies  from  12// 
to  25//  long  and  8//  to  15/*  wide.  The  protoplasm  is  finely  granular, 
excepting  for  two  rows  of  larger  granules  which  begin  on  either  side  of 
the  nucleus  and  converge  posteriorly.  From  the  anterior  part  project 
three  to  four  flagella,  which  seem  to  begin  at  a  basal  thickening  near  to  or 
connected  with  the  more  or  less  oval,  indistinctly  vesicular  nucleus.  From 
the  origin  of  the  flagella  an  undulating  membrane  extends  backward. 
The  body  also  seems  to  possess  a  certain  linear  structure  connected  with 
the  membrane.  Contractile  vacuoles  have  not  been  seen. 


COCCIDIA  497 

The  trichomonas  hominis  Davaine,  found  frequently  in  the  human 
alimentary  canal,  is  very  similar  to  the  trichomonas  vaginalis,  but  it 
is  smaller  and  more  pear-shaped.  This  form  has  been  found  often  in 
acute  diarrhoeas,  but  no  causal  relation  between  it  and  the  pathological 
process  has  been  shown. 

A  similar  form  has  been  seen  a  few  times  in  lung  gangrene,  aspira- 
tion pneumonia,  and  bronchiectasis. 

Lamblia  intestinalis  (Lambl,  1859),  a  flagellate  belonging  to  this 
group,  parasitic  in  the  small  intestines  of  mice,  rats,  rabbits,  dogs,  cats, 
and  sheep,  has  also  been  found  occasionally  in  the  human  intestines. 
It  is  beet-shaped,  bilaterally  symmetrical,  10//  to  21//  long  and  5/z 
to  12//  wide,  possessing  flagella  9//  to  14//  long.  Anteriorly  this  species 
has  a  characteristic  concavity,  the  rim  of  which  seems  to  be  contractile, 
forming  a  sucking  apparatus.  The  eight  flagella  of  the  organism  are 
arranged  in  pairs :  one  anteriorly,  two  laterally,  and  one  posteriorly.  The 
nucleus  is  situated  anteriorly  and  has  a  central  constriction.  The  pro- 
toplasm of  the  body  is  thick  and  hyaline.  Contractile  vacuoles  have  not 
been  seen.  Schaudinn  has  recently  observed  encystment,  copulation, 
and  complicated  nuclear  changes  in  this  organism. 

Infection  follows  the  ingestion  of  the  cysts  with  unclean  food.  The 
parasites  fasten  themselves  to  the  free  surfaces  of  the  epithelial  cells  by 
their  sucking  apparatus,  but  seem  to  exert  no  harmful  influence  on 
their  hosts.  They  have  been  found  most  frequently  in  poor  children 
who  play  often  in  dirt  containing  the  cysts.  Repeated  small  doses  of 
calomel  will  cause  their  disappearance  from  the  feces. 

Coccidiomorpha. 

Class:  Sporozoa. 

Subclass :  Telosporidia. 

Order :  Coccidiomorpha. 

Suborder:  Coccidia. 

Coccidium  cuniculi  (Rivolta,  1878). 

The  coccidium  cuniculi  is  a  frequent  parasite  of  the  rabbit.  Young 
rabbits  are  especially  susceptible,  and  extensive  epidemics  may  occur  in 
breeding  houses.  The  symptoms  are  fever,  diarrhoea,  yellowish  mucous 
discharge  from  the  nose  and  mouth,  and  progressive  wasting.  The 
liver  is  much  enlarged  and  shows  throughout  its  substance  variously  sized 
gray- white  tubercles,  generally  surrounded  by  a  capsule,  and  containing  a 
slimy  mass  of  degenerated  host  cells,  in  which  are  embedded  the  parasites. 
The  parasites  are  also  found  in  the  feces  and  in  the  epithelial  cells  of 
the  intestines,  gall-ducts,  and  liver.  The  acute  stage  of  the  disease 
lasts  about  three  weeks.  The  contents  of  the  coccidial  tumors  in  animals 
that  have  withstood  the  infection  may  later  be  emptied,  leaving  only  a 
mass  of  cicatricial  tissue.  In  such  animals  the  oocysts  may  remain  for 
a  long  time  in  the  gall-bladder  and  intestines,  and  by  passing  out  gradu- 
ally with  the  feces  may  provide  a  source  of  infection  for  other  animals. 
The  infection  is  carried  by  food  soiled  with  cyst-containing  feces.  The 

32 


498 


PROTOZOA 


cysts  pass  with  the  food  into  the  stomach,  where  the  cyst  wall  and  the 
spore  sack  are  destroyed  and  the  sporozoites  are  set  free.  The  motile 
sporozoites  pass  through  the  ductus  choledochus  into  the  liver,  some 
probably  passing  into  the  intestines  and  infecting  the  cells  directly, 
a  later  infection  of  the  intestines  occurring  from  forms  developed  in  the 
liver.  The  organism  develops  within  the  epithelial  cells  of  the  liver  and 
gall-ducts  until  the  cells  are  finally  broken  down  and  tissue  cysts  are 
formed,  within  which,  after  more  or  less  complicated  changes,  cysts  of 
the  parasite  are  again  formed. 


FIG. 152 


a  b  c  d  e  f  g  h  i 

Showing  spore  formation  in  coccidium  cuniculi  from  the  liver  of  the  rabbit :  a  and  b,  young  stage 
in  the  epithelial  cells  of  the  gall-ducts  (the  small  oval  is  the  cell  nucleus);  c,  d,  and  e,  the  fertilized 
oocyst ;  in  d  the  protoplasm  is  beginning  to  shrink  away  from  the  cyst  wall,  and  in  e  it  has  con- 
tracted into  a  spherical  form ;  /,  segmentation  into  four  sporoblasts  ;  g,  elongation  of  the  sporoblasts 
to  form  spores ;  h,  four  complete  spores  in  the  oocyst ;  i,  single  spore  more  highly  magnified,  show- 
ing the  two  sporozoites  and  a  small  quantity  of  residual  protoplasm.  The  life  cycle  has  been  fuUy 
worked  out  by  Simon.  (After  Balbiani,  from  Doflein.) 

A  few  cases  of  human  infection  of  the  liver  with  the  coccidium  cunic- 
uli have  been  reported.  The  coccidium  hominis  Rivolta  found  a  few 
times  in  the  human  intestines,  as  well  as  similar  coccidia  found  in  the 
intestines  of  lower  animals,  may  belong  to  the  same  species. 

Coccidium  bigeminum  (Stiles)  is  found  in  the  feces  of  dogs,  cats,  pole- 
cats, and  possibly  human  beings.  The  organism  is  characterized  by 
the  division  of  the  oocyst  into  two  united  cysts,  containing  four  spores. 
The  size  is  8ft  to  15/>«.  The  life  cycle  is  not  well  known. 

Coccidioides  Immitis  Rixford  and  Gilchrist  (1897). 

The  organism  occurring  in  certain  cases  of  skin  and  lung  infection  in 
man  and  described  under  the  above  name  was  classed  by  the  authors  with 
the  coccidia;  but  it  has  been  shown  by  Moffitt,  Ash,  and  especially  by 
Ophiils  to  be  a  mould,  its  fungous-like  characters  developing  on  the 
usual  artificial  culture  media.  The  description  given  by  Rixford  and 
Gilchrist  of  the  morphology  of  the  parasite  in  the  tissues  is  the  same 
as  that  of  the  organisms  studied  by  Ophiils.  In  each  nodule  formed  in 
the  disease  one  to  several  parasites  are  found  either  free  or  lodged 
in  a  giant  cell.  The  parasites  have  the  form  of  "rounded  protoplasmic 
masses  20/J.  to  80//  in  diameter,  surrounded  by  a  thick,  enveloping 
membrane.  Their  multiplication  is  effected  by  a  series  of  bipartitions 
which  go  on  within  the  membrane ;  the  latter  then  bursts  and  sets  free 
the  young  parasitic  elements,  which  grow  in  situ  or  are  carried  away 
by  the  blood  or  lyinph."  Among  Ophiils'  conclusions  are  the  follow- 
ing: "  The  lesions  produced  by  this  fungus  fall  under  the  general  head 


COCCIDIA  499 

of  infectious  granulomata,  and  consist  partly  in  nodules  resembling 
altogether  those  produced  by  the  tubercle  bacilli  and  partly  in  chronic 
abscesses.  The  adult  forms  of  the  parasite  are  more  apt  to  produce 
nodules,  the  sporulating  forms  abscesses.  The  fungus  is  pathogenic 
for  dogs,  rabbits,  and  guinea-pigs,  probably  other  animals  also,  and  in 
them  produces  lesions  very  similar  to  those  which  we  encounter  in  the 
human  being  in  this  disease." 


CHAPTER   XL. 

MALARIAL  PARASITOLOGY. 
Suborder:  Hsemosporidia. 

THE  causative  agent  of  malaria  is  a  protozoon  which  is  classified 
with  the  sporozoa  (suborder:  hoemosporidia).  It  is  now  universally 
acknowledged  to  be  the  sole  cause  of  what  is  properly  included  under 
the  somewhat  misleading  term  of  malaria,  a  term  which  signifies  "  bad 
air." 

The  actual  discoverer  of  the  protozoon  is  Laveran,  who  recognized 
as  early  as  1880  the  true  nature  of  the  dancing  pigment  which  long 
before  him  had  been  observed  in  intermittent  fever.  The  relationship 
of  the  paroxysm  to  the  segmentation  of  the  parasite  was  recognized 
later  by  Golgi.  Recently  the  relationship  of  certain  insects  to  malarial 
parasites  has  been  demonstrated  by  Ross,  and  has  been  abundantly 
confirmed  by  many. 

Before  entering  upon  a  description  of  these  parasites  in  general,  it 
may  be  stated  that  they  have  a  double  cycle — i.  e.,  as  a  tapeworm 
requires  two  hosts  to  complete  its  life  cycle,  so  does  the  malarial  proto- 
zoon. The  intermediate  host  in  this  case  is  a  mosquito.  At  this  day 
there  ought  to  be  no  more  skepticism  as  to  the  role  played  by  certain 
mosquitoes;  but  one  must  not  forget  that  it  is  a  special  genus,  and 
fortunately  a  relatively  rare  one,  which  causes  the  perpetuation  of 
these  protozoa.  The  characteristics  of  this  mosquito  are  such  that  even 
people  untrained  in  such  observations  can  readily  tell  the  malaria- 
carrying  mosquito  from  its  relatively  harmless  and  prolific  simile.  Hence, 
a  few  useful  points  on  the  subject  are  given  below. 

The  cycle  in  the  human  being  is  known  as  the  asexual  cycle,  or  the 
monogony ;  while  the  primary  cycle  carried  on  in  the  viscera  of  the  insect 
is  called  the  sexual  cycle,  or  amphigony.  There  is  undoubtedly  in  the 
human  being  a  third  unknown  step,  a  sort  of  hibernation  or  lying  dor- 
mant, attributable  to  a  modification  of  a  form  which  possibly  belongs 
to  the  monogony. 

In  the  asexual  cycle  the  parasite  grows  and  divides  into  segments 
which  correspond  to  the  young  form  from  which  the  parasite  originates 
without  the  influence  of  a  sexual  element.  This  process  of  apparently 
agamogenetic  division  goes  on  periodically  for  a  limited  length  of  time. 
It  seems  as  if  the  power  for  self-reproduction  is  exhausted  after  a 
time,  and  as  if  possibly  also  the  blood  of  the  host  has  formed  circu- 
lating compounds  antagonistic  to  the  parasite.  These  explanations  for 
the  time  limit  seem  reasonable. 


MALARIAL  PARASITOLOGY  501 

At  all  events  the  sporulat  ion  ceases  after  a  while,  and  there  arc  distinct 
changes  in  the  morphology  of  the  parasite.  Finally,  certain  bodies  are 
formed  which  do  not  segment,  and  it  is  these  which  will  now  be  described 
in  detail.  They  are  known  as  gametes,  or  sexual  forms,  and  represent  a 
male  and  female  element  which,  however,  cannot  conjugate  in  the  human 
blood.  When  certain  species  of  mosquitoes  imbibe  blood  containing 
such  gametes,  the  process  of  conjugation  is  carried  on  in  the  chyme 
stomach  of  the  insect.  How  this  proceeds  will  be  described  presently; 
suffice  it  to  say  here  that  cysts  are  formed  in  the  stomach  wall  of  the 
insect,  which  when  matured  discharge  an  enormous  number  of  fila- 
mentous sporozoites  into  the  body  cavity  of  the  mosquito,  whence  they 
reach  what  is  usually  called  the  salivary  gland  of  the  insect.  From  this 
gland  it  is  but  a  step  to  the  proboscis,  and  when  the  insect  thus  infected 
"bites"  a  human  being  numerous  sporozoites  pass  into  the  circulation. 
Just  how  these  sporozoites  are  transformed  into  the  well-known  young 
form  seen  on  the  red  corpuscle  is  not  known. 

While  these  processes  are  rather  fully  understood,  the  relapses  of  sup- 
posedly cured  cases  are  shrouded  in  mystery.  The  observations  tend  to 
show  that  there  is  a  difference  in  the  parasites  of  relapses  when  compared 
with  those  of  a  recent  primary  infection,  and  for  my  part  I  do  not  usually 
have  any  difficulty  in  telling  the  parasites  of  relapse  from  those  of  a  recent 
primary  infection.  Moreover,  I  have  also  found  that  the  parasites  of  a 
supposed  second  infection  resemble  those  of  a  relapse,  and  in  the  light  of 
this  observation  I  doubt  if  a  second  infection  in  the  proper  sense  of  the 
word  is  ever  possible  within  a  certain  length  of  time.  It  is  not  within  the 
scope  of  this  article  to  give  a  detailed  description  of  the  forms  which  in 
my  opinion  point  to  a  relapse ;  suffice  it  to  say  that  the  parasites  in  ques- 
tion show  a  tendency  to  remain  dwarfed,  and  show  fewer  chromatin 
bodies  and  greater  irregularities  in  division  than  those  of  what  I  term  a 
recent  primary  infection.  These  abnormalities  may  be  referable  to  the 
persistent  effect  of  antibodies.  One  form  of  parasite  is  so  constant  in 
protracted  cases  and  differs  so  much  from  the  ordinary  gamete  that  a 
special  description  will  be  given. 

The  different  varieties  of  malarial  and  kindred  parasites  in  man 
and  animals  have,  as  might  be  expected,  much  in  common.  Hence 
a  general  description  of  the  cycle  of  one  form  will  suffice  to  elucidate 
the  cycle  of  the  others. 

Generalities  of  Cycle. — A  young,  amoeboid,  colorless,  more  or  less 
rounded  parasite,  measuring  approximately  two  microns  in  diameter, 
is  seen  attached  to  a  red  corpuscle.  After  a  certain  length  of  time, 
usually  a  few  hours  later,  granules  or  rodlets  of  pigment  make  their 
appearance;  these  granules  represent  the  haemoglobin  on  which  the 
parasite  has  been  feeding,  and  it  is  now  transformed  into  what  is  com- 
monly termed  melanin.  They  will  be  seen  to  be  in  passive  motion,  the 
degree  of  motility  depending  upon  the  currents  in  the  protoplasm  in 
which  they  are  embedded,  and  they  vary  in  the  different  varieties  and 
at  different  stages  of  the  asexual  phase.  After  a  further  lapse  of  time 
this  pigment  is  seen  to  gather  centrally  and  evidences  of  cell  division 


502  PROTOZOA 

can  then  be  made  out;  there  may  be  a  distinct  rosette-like  arrangement 
or  not.  The  next  step  is  the  breaking  up  of  the  parasite  into  segments 
free  from  pigment.  The  further  observation  of  these  segments,  each 
of  which  represents  a  young  parasite,  identical  with  the  young  form 
from  which  its  progenitor  was  built  up,  is  a  matter  of  difficulty.  I  had 
the  good  fortune  of  watching  one  in  a  fresh-blood  preparation  from 
the  moment  of  its  detachment  from  the  mother  substance,  and  saw  it 
approach  corpuscle  after  corpuscle.  Its  movements  were  gregarine- 
like.  It  did  not  attach  itself  to  a  corpuscle  within  an  hour,  presumably 
because  of  the  change  in  the  nature  of  the  blood  and  its  plasma. 

In  the  blood  stream  it  is  but  rarely  seen  free  in  the  plasma,  and  it 
would  appear  that  it  rapidly  attaches  itself  to  a  neighboring  corpuscle. 
That  would  tend  to  explain  the  fact  that  in  certain  forms  of  malaria 
it  is  not  an  uncommon  thing  to  find  several  parasites  in  one  corpuscle; 
this  is  seen  especially  in  those  cases  where  segmentation  takes  place 
in  the  bone-marrow,  and  where  on  account  of  the  absence  of  the  velocity 
of  the  blood  stream  the  parasites  are  less  apt  to  be  scattered  than  in 
the  circulating  blood. 

It  is  to  be  remembered  that  the  process  of  growth  and  division  can- 
not be  readily  observed  in  all  the  forms  of  malaria  because  certain 
parasites  complete  their  asexual  cycle  in  the  viscera,  chiefly  also,  it 
seems,  in  the  bone-marrow  and  spleen. 

Three  types  of  parasites  are  recognized.  Classified  zoologically  they 
are:  Plasmodium  vivax  (tertian),  malarise  (quartan),  and  praecox 
(sestivo-auturnnal) . 

The  life  cycle  of  each  presents  certain  characteristics  which  hold  it 
apart  from  the  others.  Some  authors  suggest  a  unity  of  parasites,  but 
the  mere  fact  that  the  gametocytes  of  tertian  and  aestivo-autumnal  fever 
differ  absolutely  from  each  other  is  a  capital  reason  why  this  is  improb- 
able; there  are  many  other  striking  differences. 

A  brief  consideration  of  the  salient  facts  follows : 

Plasmodium  Vivax,  the  Parasite  of  Tertian  Ague. 

The  young  form  is  more  difficult  to  find  and  to  recognize  than  any 
of  the  other  forms.  A  pale  area  is  seen  on  an  otherwise  unaltered  red 
corpuscle,  situated  usually  eccentrically,  about  one-tenth  the  size  of 
the  red  corpuscle  or  about  one-fourth  its  diameter,  when  at  rest  pre- 
senting a  rounded  appearance,  usually  actively  amoeboid,  throwing  out 
pseudopodia  which  are  distinct,  never  remaining  long  in  the  same  focal 
plane,  frequently  dipping,  so  to  speak,  into  the  substance  of  the  cor- 
puscle. It  is  often  called  the  hyaline  form  because  it  is  free  from 
pigment,  but  it  is  not  hyaline  in  the  proper  sense  of  the  term.  It  is 
also  called  the  ring  form,  because  of  its  resemblance  to  a  ring  in  stained 
preparations;  but  it  is  never  a  true  ring. 

The  forms  intermediate  between  this  and  the  segmentation  stage 
are  simply  larger  parasites,  which  are  readily  found  on  account  of  the 
pigment  granules  which  they  contain. 


MALARIAL  PARASITOLOGY  508 

In  the  earlier  stages  they  resemble  the  younger  form  except  that  the 
pseudopodia  appear  to  be  exaggerated;  besides  there  is  a  rapid  dancing 
of  the  pigment  due  to  protoplasmic  currents  in  the  parasite.  The  infected 
corpuscle  is  swollen  and  paler.  Later  the  parasite  occupies  the  greater 
portion  of  the  corpuscle,  which  is  now  more  difficult  to  make  out.  The 
pigment  is  still  more  evident,  so  that  this  form  is  therefore  most  readily 
found. 

The  anatomical  changes  which  have  been  going  on  in  the  parasite 
are  best  studied  in  properly  stained  smear  preparations.  They  become 
presently  sufficiently  distinct  to  be  followed  in  the  living  parasite;  the 
pigment  gathers  more  or  less  centrally  into  a  compact  mass,  and  a  per- 
ipheral notching  indicates  that  the  parasite  is  preparing  to  divide  into  a 
number  of  segments;  the  number  of  segments  varies  even  in  perfectly 
typical  acute  cases,  but  from  twelve  to  twenty  may  be  counted  as  soon 
as  the  parasite  has  become  fully  mature.  Suddenly  these  spheroidal 
segments  separate  from  each  other;  a  corpuscular  remnant  and  the 
pigment  float  away  and  are  ultimately  carried  to  the  spleen.  Phago- 
cytic  cells  ingest  these  pigment  masses.  The  young  parasites  attach 
themselves  to  red  corpuscles  as  already  stated.  The  mature  tertian 
parasite  is  usually  larger  than  a  red  corpuscle. 

The  varying  number  of  segments  is  possibly  accounted  for  by  the 
difference  in  size,  age,  and  constitution  of  the  infested  corpuscles.  Later, 
when  the  blood  may  have  formed  antibodies,  irregularities  are  still  more 
readily  explained.  If  suitable  smears  are  made  from  blood  containing 
such  tertian  organisms  the  life  cycle  can  be  studied  much  more  closely. 

The  making  of  these  smears  is  a  simple  matter.  There  are  the  cover- 
glass  and  the  slide  methods,  both  of  which  have  their  peculiar  advan- 
tages. To  make  a  cover-glass  preparation,  two  square,  very  thin,  hence 
flexible,  cover-glasses  are  cleaned.  Holding  one  with  thumb  and  index 
fingers  by  opposite  corners  the  tip  of  a  drop  of  blood  obtained  by  needle 
puncture  of  finger  or  lobe  of  ear  is  made  to  touch  the  centre  of  the  cover- 
glass,  and  the  second  clean  cover-glass  held  similarly  is  allowed  to  fall 
upon  the  first  one  in  such  a  manner  that  the  corners  do  not  coincide. 
The  blood  droplet  spreads  by  capillarity  into  a  thin  film,  which  is  a 
sign  to  pull  the  two  covers  apart  in  the  plane  in  which  they  lie;  good 
results  depend  upon  cleanliness,  rapidity,  and  success  in  sliding  the 
two  covers  apart. 

A  simpler  way  is  to  polish  two  slides.  The  tip  of  the  exuded  blood 
drop  is  made  to  touch  one  slide  near  one  end  and  the  edge  of  the  second 
slide  held  at  an  acute  angle  to  the  first  one  is  made  to  bisect  the  drop, 
which  will  spread  at  the  point  of  contact  by  capillarity  across  the  slide. 
Upon  pulling  the  second  or  spreading  slide  over  the  first  slide,  never 
changing  the  angle  and  applying  gentle  pressure,  a  thin  layer  of  blood 
suitable  for  examination  will  be  formed.  A  slide  made  in  this  manner 
should  be  dried  immediately  by  agitation  in  the  air.  It  may  then  be 
fixed  and  stained  in  various  ways.  I  would  recommend  a  modification 
of  a  method  devised  by  me  in  1900,  which  is  given  here  below,  together 
with  some  others. 


504  PROTOZOA 

In  a  suitably  stained  preparation  (using  a  chromatin  dye)  the  young 
parasite  appears  to  be  a  disk  consisting  of  a  basic  (blue)  periphery,  the 
body,  including  a  metachromatically  stained,  rounded,  compact  (red) 
chromatin  body,  often  called  the  nucleus,  which  tends  to  give  the  para- 
site the  form  of  a  signet  ring,  and  of  a  central,  pale,  unstained  area,  known 
as  the  achromatic  zone. 

Later  stages  up  to  a  certain  number  of  hours  show  simply  changes  in 
size  and  outline  of  the  blue-stained  body.  Then  the  red  chromatin  body 
loosens  up  and  presently  its  substance  divides  into  an  increasing  number 
of  angular  pieces,  which  are  the  first  evidence  of  regeneration.  By  the 
time  that  chromatin  division  is  completed  the  angular  chromatin  masses 
will  have  assumed  a  rounded  form,  and  will  be  seen  to  exhibit  ultimately 
the  same  strong  affinity  for  certain  dyes  which  is  seen  in  the  compact 
chromatin  body  of  the  young  ring-like  form.  At  this  stage  the  here- 
tofore scattered  pigment  appears  in  one  clump.  Good  technique  will 
always  show  a  corpuscular  remnant  even  at  this  time.  The  achromatic 
zone  mentioned  will  be  seen  to  develop  with  the  chromatin,  and  when 
the  next  step,  namely,  the  division  of  the  body  of  the  parasite,  is  seen 
to  be  completed,  there  will  be  as  many  achromatic  bodies  as  there  are 
chromatin  bodies,  each  having  an  equal  share  of  the  basic  mother-body, 
each  representing  a  young  parasite.  These  young  parasites  next  escape 
from  their  envelope  or  whatever  substance  may  have  held  them  together, 
are  set  free  in  the  plasma,  and  attack  without  delay  the  corpuscles. 

It  is  an  open  question  as  to  whether  the  parasite  sits  on  or  lives  in 
the  corpuscles.  The  latter  supposition  appears  to  be  more  plausible, 
especially  because  the  segmenting  form  retains  to  the  last  a  corpuscular 
remnant  and  because  the  young  amoeboid  form  may  be  seen  to  dip  into 
the  corpuscle,  leaving  one  focal  plane  and  appearing  in  a  deeper  one. 
How  could  that  be  possible  if  it  simply  adhered  to  the  surface?  The 

DESCRIPTION  OF  PLATE  II. 

(The  numbers  are  placed  immediately  above  the  respective  form.) 
1  to  4.  Young  forms ;  unpigmented. 

5  to  8.  Gradual  pigmentation  and  growth  of  the  parasite ;  the  chromatin  is  loosening  up. 
9  to  13.  Active  division  of  chromatin  from  two  into  twelve  or  more  pieces,  which  are  at  first 
angular,  but  become  rounded. 

14  to  15.  Complete  segmentation  ;  collection  of  pigment  in  single  lump. 

16.  Bursting  of  segmenting  parasite  and  liberation  of  young  forms,  each  of  which  de  novo  infects 
and  destroys  a  new  corpuscle. 
17  to  19.  Young  male  sexual  forms. 

20.  Male  sexual  forms  (microgametocyte).  The  coiled-up,  centrally  situated  chromatin  fibrils  are 
the  flagella  or  microgametes. 

21  to  22.  Female  sexual  form  ;  stain  deeper  than  in  male  ;  less  chromatin,  situated  peripherally  ; 
often  extracorpuscular ;  macrogamete.  (These  gametes  are  the  analogues  of  the  crescentic  bodies 
of  sestivo-autumnal  malaria.) 

23.  Abnormally  situated  accessory  chromatin  body. 

24.  Two  parasites  maturing  side  by  side. 

25  to  26.  Effect  of  quinine  on  body  of  parasite. 

27.  Resistant  form  capable  of  causing  relapse  (?). 

28.  Abnormal  segmentation  of  immature  parasite. 

The  various  oval  and  rounded  bodies  are  blood  platelets.  The  red  blood  corpuscles  are  simply  out- 
lined ;  their  color  and  degree  of  degeneration  when  infected  will  depend  entirely  upon  the  technique. 
The  body  of  the  parasite  is  blue;  the  chromatin  body  is  carmine ;  the  pigment  is  simply  shown  in 
black. 


PLATE   II. 


Various  Stages  in  the  Development  of  the  Malarial  Parasite. 
Tertian  type.     (Goldhorn.^ 


OF  THF 

UNIVERSITY 

<*r  iroK]^ 


MALARIAL  PARASITOLOGY  505 

denser  protoplasmic  boundary  zone  of  the  corpuscle  offers  no  resistance 
to  the  parasite. 

Tlu'  technique  recommended  below  shows  that  the  infested  cor- 
puscle early  undergoes  a  granular  degeneration,  which,  curiously  enough, 
in  the  first  few  hours  resembles  the  ordinary  granular  stroma  degenera- 
tion with  basic  affinity,  while  it  is  later  seen  that  the  affinity  of  the  then 
more  numerous  granules  is  more  acid  or  at  least  the  staining  is  no  longer 
orthochromatic,  the  blue  being  superimposed  by  a  red;  in  other  words, 
these  granules  stain  later  metachromatically.  The  greater  the  loss  or 
transformation  of  the  haemoglobin  the  greater  the  number  of  granules. 
This  holds  good  only  for  tertian  parasites,  the  sestivo-autumnal  variety 
causing  practically  no  appreciable  change  though  the  same  technique 
be  used. 

But  the  mere  recognition  of  the  malarial  parasite  in  the  blood  is 
insufficient  to-day.  Not  only  should  the  physician  know  the  variety  with 
which  he  is  dealing,  but  also  it  will  be  to  his  advantage  to  know  some- 
thing about  the  progress  which  the  disease  is  making  by  study  of  the  circu- 
lating parasite.  This  will  seem  difficult  at  first,  but  a  relatively  small 
amount  of  study  will  show  the  value  of  the  recognition  of  certain  changes. 

In  tertian  fever  much  can  be  learned  from  the  study  of,  let  us  say,  three 
typical  cases,  viz. :  (1)  a  recent  primary  infection;  (2)  a  primary  infection, 
but  of  long  standing;  (3)  a  relapse.  The  student  who  knows  the  forms 
occurring  in  the  typical  recent  primary  infection  will  readily  observe  many 
morphological  differences  between  these  and  the  parasite  of  the  pro- 
tracted case.  He  will  then  also  find  at  least  two  forms  which  are  not 
readily  classified  with  those  of  his  recent  primary  infection.  These 
forms  are  the  gametocytes  or  gametes,  the  analogues  of  the  crescent. 

The  microgametocyte  of  tertian  malaria  is  a  large  parasite  with  little 
affinity  for  methylene  blue,  carrying  more  conspicuous  pigment  arranged 
frequently  as  a  wreath  around  a  large  achromatic  zone  in  which  filaments 
of  chromatin  lie.  These  filaments  are  the  microgametes,  also  known 
as  the  flagella.  When  living  the  pigment  of  this  parasite  is  immotile 
until  just  before  the  parasite  flagellates,  at  which  time  an  unusual 
activity  of  the  granules  is  seen. 

The  second  form  is  the  macrogamete,  which  is  an  extracorpuscular 
body,  staining  deeply  in  methylene  blue,  having  a  hazy  chromatin 
mass  in  indistinct  achromatic  zone  usually  situated  peripherally. 

The  young  form  which  grows  ultimately  into  a  microgametocyte  is 
ring-shaped,  with  a  heavy  chromatin  body  well  within  the  achromatic 
zone. 

In  relapses  a  third  form  is  met  with  which  has  a  compact  chromatin 
body,  surrounded  by  a  paler  area,  that  can,  however,  be  stained  pinkish 
by  chromatin  dyes."  The  recognition  of  these  forms  is  of  the  utmost 
importance  because  the  prognosis  depends  upon  them.  They  are  quite 
resistant  to  quinine  and  other  drugs,  and  it  appears  as  if  cases  in 
which  these  forms  are  seen  are  much  more  prone  to  relapse  than 
promptly  treated  recent  primary  infections. 

Also  the  absence  of    gametocytes  from   the  blood   means  that  no 


506  PROTOZOA 

amount  of  such  blood  that  anopheles  mosquitoes  may  imbibe  can  ever 
infect  the  mosquito. 

When  studying  old  cases  and  relapses  it  will  be  seen  that  the  char- 
acteristics of  the  three  varieties  are  lost  to  a  certain  extent.  But  other 
factors  will  have  changed  accordingly;  the  paroxysms  will  have  become 
irregular,  protracted,  less  severe,  and  will  eventually  occur  widely  apart. 
Now,  as  the  paroxysm  depends  upon  the  segmentation  of  the  protozoon 
or  the  probable  liberation  of  a  toxin,  any  changes  in  the  morphology 
might  be  expected  to  influence  the  periodicity  and  character  of  the 
paroxysm,  and  such  is  actually  seen  to  be  the  case. 

An  abnormal  temperature  chart  can  usually  be  explained  by  proper 
study  of  the  forms  of  parasites  present.  If  quinine  has  been  admin- 
istered it  is  to  be  considered,  as  its  effect  on  the  morphology  of  the 
parasite  is  profound  and  rapid.  The  part  most  and  first  affected  is  the 
blue  staining  body;  later  follow  eccentricities  of  the  chromatin,  such 
as  multiple  bodies  in  young  forms,  extension  of  chromatin  bodies,  and 
dwarfing,  just  such  changes  as  might  have  occurred  in  time  if  the  body 
had  been  allowed  to  combat  the  parasite  without  the  aid  of  drugs. 

The  notion  that  the  parasites  can  be  found  only  at  the  time  of  the 
paroxysm  is  still  in  the  minds  of  many;  it  is  erroneous. 


The  .ffistivo-autumnal  Parasite. 

In  a  recent  primary  infection  only  young  forms  can  be  found  in  the 
peripheral  blood;  their  pigment  is  so  scanty  as  to  render  their  recog- 
nition in  fresh  blood  a  matter  of  experience;  dancing  pigment  is  prac- 
tically never  seen  in  such  cases. 

Later  in  the  disease  the  gametocytes  develop;  they  are  absolutely 
characteristic,  readily  found,  and  of  diagnostic  value. 

In  the  fresh  blood  the  young  parasite  is  seen  as  a  fairly  sharply  de- 
fined, rounded,  slightly  amoeboid  body;  the  infected  corpuscle  may  be 
crenated;  it  may  have  what  has  been  termed  a  "brassy"  appearance. 

Such  forms  stained  show  extremely  thin  and  small  rings,  with  one 
or  more  chromatin  bodies,  situated  not  infrequently  within  the  achro- 
matic zone.  There  are  frequently  several  parasites  in  one  corpuscle; 
five,  six,  and  seven  have  been  seen.  Later  the  parasite  shows  a  few 
heavy,  blackish,  pigment  granules,  and  an  increase  in  the  size  of  the  body 
is  seen.  It  is  very  exceptionally  that  one  finds  in  the  peripheral  blood 
forms  approaching  segmentation  or  actually  segmenting.  The  various 
steps  of  chromatin  division  seem  to  take  place  chiefly  in  the  bone- 
marrow  and  the  viscera.  The  parasite  does  not  ordinarily  attain  the 
size  of  the  tertian  form,  and  there  are  usually  fewer  segments.  Their 
arrangement  is  apt  to  be  very  symmetrical. 

Upon  developing  the  gametocytes  these  are  seen  at  times  in  large 
numbers.  They  are  first  ovoidal  and  later  crescentic.  The  blackish 
pigment  lies  in  a  wreath  around  the  thus  hidden  chromatin  filaments. 
There  may  be  a  corpuscular  remnant  or  not.  There  seems  to  be  a  sort 


MALARIAL  PARASITOLOGY 


507 


of  capsule  at  times.  The  ends  stain  more  deeply  in  methylene  blue  than 
the  rest  of  the  parasite,  a  phenomenon  which  is  known  as  polar  staining. 
The  pigment  is  quiescent  in  the  fresh  specimen.  Upon  prolonged 
observation,  say  twenty  minutes  or  less,  according  to  temperature,  etc., 
these  crescentic  bodies  are  transformed  into  spherical  bodies;  the  pigment 
of  certain  ones  of  these  becomes  actively  motile,  due  to  internal  agita- 
tion of  the  chromatin  fibrils,  which  will  presently  emerge  as  flagella. 
Their  movements  are  very  rapid,  corpuscles  are  knocked  about,  and 
finally  these  flagella,  which  represent  the  male  sexual  element,  the 
spermatozoid,  so  to  speak,  become  detached  and  go  in  search  of  the 
female  element.  In  birds  one  may  actually  observe  the  process  of 
conjugation  in  slide  preparations  even  without  the  aid  of  a  moist  cham- 
ber and  heat.  This  transformation  of  crescentic  bodies  never  occurs 
in  the  human  blood.  It  will  be  seen  that  it  belongs  to  the  sexual  cycle 
which  occurs  in  the  stomach  of  the  mosquito. 


FIG.  153 


©  O 


.Estivo-autumnal  type  :  1  to  7,  various  young  non-pigmented  forms  ;  8  to  10,  larger  forms  from 
peripheral  blood  ;  11  to  14,  various  ovoid  bodies  ;  15  to  17,  crescentic  bodies ;  18  to  19,  crescentic 
bodies  after  removal  of  pigment  showing  chromatin  filaments  in  achromatic  zone ;  20  to  21,  abnor- 
mal forms. 

Crescents  do  not,  in  my  opinion,  ever  divide,  either  agamogenetically 
or  otherwise,  in  the  blood.  Forms  which  show  a  constriction  are  rather 
to  be  referred  to  a  twin  maturation.  Nor  do  I  believe  that  the  chro- 
matin of  microgametocytes  ever  divides  by  segmentation;  it  seems  to 
me  that  the  coiled-up  filaments  are  rapidly  rearranged  and  then  indi- 
vidually extended  as  so  many  flagella  or  microgametes. 

In  fatal  cases  the  formation  of  crescents  may  not  take  place;  tin- 
blood  infection  with  young  parasites  is  then  enormous,  every  field  of 
the  microscope  showing  numbers  of  them. 

In  the  study  of  a?stivo-autumnal  fever  it  is  to  be  remembered  that 
crescents  when  found  indicate  that  the  disease  is  of  some  standing,  for 
such  sexual  forms  (gametes)  are  not  formed  until  the  asexual  propaga- 
tion is  waning.  The  recognition  of  these  ovoidal  and  crescentic  bodies 
is  easy.  But  as  there  are  no  readily  discoverable  pigmentecl  forms  in 


508  PROTOZOA 

the  peripheral  blood  in  the  early  stages,  it  is  necessary  to  be  thoroughly 
familiar  with  the  young  aestivo-autumnal  forms.  Chromatin  staining  for 
them  cannot  be  too  much  recommended,  as  there  is  little  that  is  charac- 
teristic about  them  when  they  have  been  stained  with  methylene  blue 
alone.  Many  a  serious  error  has  been  made  by  adhering  to  the  an- 
tiquated idea  that  parasites  should  be  looked  for  in  the  fresh  blood, 
as  these  young,  non-pigmented,  so-called  hyaline  forms  cannot  be  readily 
recognized  by  the  inexperienced,  while  it  is  an  easy  matter  to  know 
and  classify  them  when  properly  stained. 

The  Quartan  Parasite. 

The  recognition  of  the  quartan  parasite  in  its  early  stages  in  the 
fresh  blood  is  not  as  difficult  as  that  of  the  tertian  form,  but  in  stained 
preparations  it  is  often  indistinguishable  from  the  latter.  The  living 
amoeboid  young  form  is  more  refractive  than  the  young  living  tertian 
form,  more  like  the  sestivo-autumnal  form,  and  it  is  just  as  sluggish  in 
its  movements.  Here,  too,  the  corpuscle  is  often  shrunken  and  looks  as 
if  it  contained  more  haemoglobin  than  in  the  case  of  infection  with  the 
tertian  parasite. 

The  growing  parasite  shows  fewer  pigment  granules  than  the  corre- 
sponding tertian  one,  and  it  is  apt  to  form  a  band  across  the  infected  cor- 
puscle. Segments  are  few  in  number,  as  a  rule,  and  the  parasite  remains 


DESCRIPTION  OF  PLATE  III. 

1.  Typical  young  tertian  form ;  the  corpuscle  shows  incipient  degeneration  ;   corpuscle  to  left 
above  shows  a  blood  platelet. 

2.  Abnormal  young  form,  showing  small  accessory  chromatin  body. 

3.  Two  parasites ;  one  a  normal  young  form;  the  second  large  form  in  crenated  corpuscle  is  an 
unusual  abnormal  form  with  very  large  achromatic  area. 

4.  5,  6.  ^Estivo-autumnal   parasites;   single,   double,   and  triple   infection;    central   elongated 
chromatin  bodies.    These  forms  are  about  the  largest  usually  seen  in  the  peripheral  blood  ;  no 
degeneration  of  corpuscle. 

7.  Tertian  parasite,  about  ten  hours  old ;  marked  degeneration  of  corpuscle. 

8.  Double  infection  of  ff  corpuscle  in  tertian  fever ;  marked  degeneration  of  corpuscle. 

9, 10, 11.  Large  tertian  parasites  showing  division  of  chromatin  previous  to  segmentation. 
12  and  14.  Complete  segmentation  of  tertian  parasite. 

13.  Double  infection  of  corpuscle,  one  parasite  reaching  maturity,  but  showing  unusually  small 
segments  ;  the  second  one  atrophied. 

15.  Tertian  parasite,  old  case  ;  while  the  parasite  is  only  half-grown,  the  chromatin  has  split  into 
several  compact  masses.    Degeneration  of  infected  corpuscle. 

16.  Dwarfed  tertian  parasite,  smaller  than  a  red  corpuscle,  but  showing  five  compact  chromatin 
bodies ;  resemblance  to  quartan  rosette. 

17.  Microgametocyte  of  tertian  malaria ;  prominence  of  blackish  pigment  surrounding  a  large 
achromatic  zone  in  which  the  microgametes  lie  coiled  up. 

18.  Tertian  macrogametes. 

19  to  23.  Crescentic  bodies  of  aestivo-autumnal  malaria. 

19.  Typical  gamete ;  pigment  surrounding  achromatic  area ;  no  chromatin  shown;  the  "bib"  is 
present.    (Male?) 

20.  Semiovoid  gamete.    (Female?) 

21.  Pigment  removed.    Elliptical  achromatic  area  in  which  the  microgametes  are  seen. 
22  and  23.  Pigment  removed  ;  chromatin  more  compact ;  possibly  female  elements. 

24.  From  a  case  of  pernicious  malaria  with  rich  infection  ;  only  hyaline  forms  in  peripheral  blood. 
Below  a  large  blood-platelet. 

NOTK.— As  the  amplification  is  not  uniform  a  comparison  of  the  parasites  with  the  blood  corpuscles 
shown  should  be  made  in  order  to  have  a  correct  conception  of  their  size. 


PLATE   III. 


Photographs  of  Tertian  and  /Estivo-autumnal  Malarial 
Parasites  in  Different  Stages  of  Develop- 
ment.     (Goldhorn.) 


M.  \L.\U1.\L  PARASITOLOGY  509 

dwarfed.  It  never  attains  the  size  of  the  tertian  form.  All  the  steps 
leading  to  completed  segmentation  are  seen  in  the  peripheral  blood. 

The  gametes  are  not  well  known.    Cases  of  quartan  are  relatively  rare. 

General  Observations.—  It  should  be  remembered  that  there  'i>  no 
quotidian  form  in  this  country.  Quotidian  paroxysms  are  either  a  double 
tertian  or  a  triple  quartan  infection. 

It  is  also  interesting  to  note  that  the  fever  curve  Incomes  atypical 
even  in  those  cases  in  which  no  quinine  has  been  given.  The  excur- 
sions will  be  less,  but  the  temperature  will  stay  up  longer  and  drop 
more  gradually;  or  there  may  be  two  consecutive  paroxysms  usually 
varying  in  intensity.  The  parasitic  forms  in  the  blood  will  vary  accord- 
ingly- 


FIG.  154 


24  ™  27 

Quartan  type  :  22  to  24.  young  forms  ;  25  to  26,  tendency  to  band  formation  ;  27  to  29,  segmenta- 
tion.   (All  forms  drawn  from  stained  smears.) 

The  relationship  between  segmentation  and  paroxysm  is  always 
noted  in  tertian  cases,  and  it  is  reasonable  to  suppose  that  the  occurrence 
of  the  paroxysm  is  referable  entirely  to  the  liberation  of  toxic  substances 
resulting  from  metabolic  activity  of  the  parasite  within  the  corpuscle. 
That  there  should  be  a  toxic  product  seems  highly  probable,  and  its 
amount  must  be  considerable  in  heavy  infections.  Cases  showing  an 
infection  of  1  to  5  per  cent,  of  all  corpuscles  are  not  infrequent;  the 
destruction  of  from  50,000  to  200,000  or  more  corpuscles  per  cubic 
millimetre  of  blood  leads  to  the  rapid  deglobularization  of  the  blood; 
hence  the  deficiency  in  numbers;  add  to  this  the  effects  of  the  meta- 
bolic products,  and  little  is  left  to  the  imagination  to  explain  the 
pronounced  anremia.  Furthermore,  the  corpuscular  remnants  will  be 
largely  carried  to  the  spleen;  hence  its  hyperplastic  condition  and 
pigmentation. 

Malarial  parasites  can  always  readily  be  found  in  recent  primary 
infections,  and  it  is  usually  only  in  old  cases  that  the  search  becomes 
difficult;  one  is,  however,  generally  rewarded  by  finding  them  if  one 
looks  long  enough  for  them. 

A  helpful  sign  is  the  finding  of  pigment  in  mononuclear  leukocytes, 
which  are  seen  about  the  time  of  a  chill,  or  to  the  period  symptom- 
atically  corresponding  to  it. 

Free  pigment  cannot  be  used  as  a  means  of  diagnosis,  as  it  may  be 
impossible  to  tell  it  from  dirt  or  dust. 

A  small  dose  of  quinine  may  drive  all  parasites  of  the  monogony  out  of 
the  peripheral  circulation;  at  all  events,  the  finding  of  them  becomes,  in 
the  absence  of  gametocytes,  a  matter  of  time  and  experience,  especially 
also  as  they  may  be  much  altered  in  appearance. 

Immunity  from  malaria  appears  to  exist  as  natural  and  acquired 
immunity.  Whether  the  usual  assertion  that  contraction  of  disease 
by  pyogenic  micro-organisms  depends  upon  a  lowering  of  the  resist- 


510 


PROTOZOA 


ance  of  the  body  holds  good  in  the  case  of  these  protozoa  is  uncertain. 
Studies  made  by  me  on  over  fifty  birds  infected  with  proteosoma  showed 
that  (1)  young  birds  invariably  became  infected,  while  old  ones  rarely 
did ;  (2)  objective  symptoms  in  young  birds  were  much  more  pronounced 
than  in  previously  healthy  older  birds;  (3)  old  birds  whose  resistance 
appeared  to  be  lowered  by  disease  were  readily  infected ;  (4)  reinfection 
of  all  birds  appeared  to  be  negative. 

The  Malaria-carrying  Mosquito  (Anopheles). — Only  generalities  can 
here  be  considered.  The  common  mosquito,  often  day-flying,  belongs 
to  the  culicidae;  it  cannot  carry  human  malaria.  It  is  distinguished 

FIG. 155 


Culex. 


Anopheles. 


Anopheles. 


Culex. 


(From  Doflein.) 


from  its  night-flying  or  dusk-flying  relative  by  its  assuming  a  different 
posture  on  the  perpendicular  wall.  While  the  culex  holds  the  body 
more  or  less  parallel  with  the  surface,  the  body  of  the  anopheles  stands 
off  at  a  marked  angle. 

Wings  of  culicidae  are  unspotted;  those  of  Anopheles  macullpennis 
and  Anopheles  punctipennis  are  spotted. 

The  proboscis  of  anopheles  points  toward  the  resting  surface,  while 
that  of  culex  does  not  do  so. 

Anopheles  varieties  bite  usually  in  the  early  evening,  while  those 
of  culex  bite  almost  at  any  hour.  * 


MALARIAL  PARASITOLOGY 


511 


The  male  mosquito  is  readily  told  from  the  female  by  its  plumed 
antennae,  those  of  the  female  being  inconspicuous. 

The  Cycle  in  the  Mosquito. — If  an  ordinary  mosquito  (culex)  is  allowed 
to  imbibe  the  blood  of  a  malarial  patient  whose  blood  shows  gameto- 
cytes  there  will  be  simply  a  digestion  of  such  blood  in  the  mosquito, 
but  no  anatomical  changes.  If,  however,  an  anopheles  mosquito 
ingests  such  blood,  immediate  changes  follow.  It  should  be  remembered 
that  only  female  mosquitoes  bite;  hence,  they  alone  can  be  responsible 
for  the  spreading  of  the  disease. 

The  flagellation  of  the  male  parasite  described  above  will  promptly 
take  place;  the  free  flagella  conjugate  with  the  female  element  in  a 
manner  comparable  to  the  impregnation  of  the  ovum  of  higher  animals 
by  spermatozoids. 


FIG. 156 


Schema  of  double  cycle. 
L.  B.  Goldhorn,  fee. 
2904. 


rest-body  in 

wall  of  stomach. 


\O6kinet  which  penetrates 
\$)lining  of  ttomach-wall 

Mosquito 


of  mosquito. 


circle-asexual  reproduction  : 
chninber  observation  shows  no  flagellation. 
Outer  circle-formation  of  (sexual)  gametes; 
moist-chamber  observation  shows  flagellation. 


Infection  of  man  through  gastro-intestinal  and 
respiratory  tract,  the  infected  mosquito  dying 
in  water,  drying  in  air  or  sucking  plant-juices, 
infecting  fresh  vegetables  (theoretical) 


This  product  of  conjugation  remains  for  a  number  of  hours  in  the 
juices  of  the  chyme  stomach  of  the  mosquito,  changing  gradually  from 
a  spherical,  immotile  body  into  an  elongated  wormlet  endowed  with 
motility.  This  penetrates  the  epithelial  lining  of  the  stomach  and 
rests  in  the  tissues;  here  it  changes  into  an  oval,  then  into  a  round  body, 
which  grows  in  the  course  of  the  next  few  days  enormously,  forming  a 
cyst  which  projects  into  the  body  cavity.  Meanwhile  the  chromatin 
will  have  been  very  active.  It  will  have  divided  into  numerous  nuclei, 
which  become  arranged  around  inactive  portions,  and  filamentous 
sporozoites  develop  from  this  chromatin.  These  sporozoites  ultimately 
fill  the  cysts,  which  rupture,  setting  them  free  into  the  cavity  of  the 
mosquito's  body;  they  then  make  their  way  to  a  glandular  structure  in 
the  thoracic  cavity  of  the  insect,  the  so-called  salivary  gland,  which  in 
turn  is  in  immediate  connection  with  the  biting  and  sucking  apparatus. 
If  now  such  an  infected  mosquito  "bites"  a  human  being  the  lubri- 


512  PROTOZOA 

eating  fluid  of  the  puncturing  apparatus  will  carry  sporozoites  into 
the  blood.  The  stages  of  development  in  the  mosquito  require  about 
seven  days. 

Staining  Methods.  EHRLICH'S  TRIACID  STAIN. — Unsuitable  for 
demonstration  of  malarial  parasites. 

JENNER'S  STAIN. — Clear  pictures  of  parasites,  which,  however, 
show  no  chromatin;  hence  unsuitable  for  study  of  finer  differential 
points. 

NocHT-RoMANOWSKY  METHOD. — Very  suitable,  but  requires  accu- 
rate mixture  of  several  fluids  just  before  using,  which  then  have  to  be 
thrown  away. 

WRIGHT'S  STAIN. — Practically  identical  with  Goldhorn's  one-solu- 
tion stain  (vide  infra),  but  less  rapid;  powerful  chromatin  stain  and 
general  blood  stain. 

POLYCHROME  METHYLENE  BLUE  (GOLDHORN). — To  prepare  the  stain 
dissolve  1  gram  lithium  carbonate  in  200  c.c.  clean  water  and  add  1 
gram  methylene  blue.  Shake  and  dissolve.  Pour  into  porcelain  dish 
over  water-bath,  stirring  frequently  until  blue  color  changes  to  a  rich 
purple.  Run  through  cotton  in  funnel;  make  up  to  200  c.c.  To  100 
c.c.  add  5  per  cent,  acetic  acid  until  a  faint  pink  is  just  visible  on  litmus 
paper  above  level  of  point  discolored  by  the  dye.  Now  add  the  remain- 
ing 100  c.c.  of  dye  and  allow  to  stand  in  open  dish  for  forty-eight  hours. 
Run  once  more  through  cotton  into  clean  bottle. 

I  have  not  found  it  necessary  to  use  distilled  water,  and  have  obtained 
satisfactory  results  with  all  the  different  forms  of  methylene  blue  I 
have  been  able  to  obtain  during  the  past  five  years.  I  now  prefer 
B-X  Gruebler. 

Fix  the  smear  by  immersion  in  commercial  wood  alcohol  for  fifteen 
to  thirty  seconds;  wash  well  and  stain  for  about  ten  to  fifteen  seconds 
in  polychrome;  wash  and  stain  for  from  fifteen  to  sixty  seconds  in  -^g- 
per  cent,  aqueous  eosin.  Wash  again  in  water  and  dry  in  air  without 
heat.  Body  of  parasites  blue;  chromatin  is  red  to  purple. 

Results  may  be  varied  by  using  polychrome  or  eosin  for  different 
lengths  of  time.  Admirable  preparations  may  be  obtained,  even  when 
there  is  precipitation,  by  just  rinsing  the  smear  a  little  in  50  per  cent, 
ethyl  alcohol.  This  will  remove  any  precipitation. 

The  simplest  method  of  staining  the  parasite  is  probably  the  follow- 
ing, recommended  by  me  for  the  staining  of  mast-cells :  Saturate  wood 
alcohol  with  methylene  blue.  Pour  on  dry  smear  for  five  to  ten  seconds 
and  wash  in  water.  Parasite  blue. 

GOLDHORN'S  ONE-SOLUTION  STAIN. — To  Goldhorn's  polychrome 
methylene  blue  (vide  supra)  add  weak,  watery  (•£•  to  YV  per  cent.)  eosin 
until  the  filtrate  is  of  a  pale-blue  color;  the  exact  amount  of  eosin  will 
depend  upon  the  degree  of  alkalinity  of  the  polychrome  and  upon  the 
amount  of  unaltered  methylene  blue  in  the  polychrome. 

The  precipitate  is  washed  with  water  and  dried  without  heat  and 
protected  from  dust.  When  absolutely  dry  it  is  dissolved  in  commer- 
cial wood  alcohol,  making  a  1  to  2  per  cent,  solution. 


MALARIAL  PARASITOLOGY  513 

The  smear  is  dried  without  heat  and  held  for  a  second  or  two  in 
the  dye.  It  is  then  dipped  slowly  into  a  vessel  with  clean  water,  film 
Me  down;  it  should  not  be  plunged  into  the  water.  The  staining 
depends  upon  the  interaction  of  the  water  with  the  film  of  dye  adhering 
to  the  blood.  Hold  preparation  in  the  water  for  a  few  seconds,  then 
move  it  about  for  a  moment,  and  rinse  in  clear  water;  clean  lower  side 
of  the  slide,  as  precipitation  will  have  taken  place  here;  hence,  do  not 
introduce  into  water  with  film  side  up.  Dipping  the  preparation  for 
a  moment  into  50  per  cent,  ethyl  alcohol  removes  smudges  and  pre- 
cipitate. 

Mode  of  Infection. — While  it  is  probable  that  infection  with  malarial 
sporozoites  occurs  in  the  great  majority  of  cases  by  the  bite  of  an 
infected  mosquito,  there  seem  to  be  cases  in  which  the  infection  has 
taken  place  in  some  other  way.  Mosquitoes  normally  feed  on  plant 
juices;  to  obtain  their  food  they  insert  their  proboscis  into  the  plant 
tissues.  Is  it  then  unreasonable  to  suppose  that  such  a  plant  would  be 
infected  in  a  manner  analogous  to  the  manner  in  which  the  human 
blood  is  infected?  Why  should  not  such  a  vegetable  eaten  uncooked 
cause  an  infection  through  the  gastrointestinal  tract?  Again,  infected 
mosquitoes  after  ovipositing  may  die  in  the  water,  and,  partaking  of 
such  water,  might  lead  to  infection;  or  if  the  insect  died  in  the  open  air 
the  sporozoites  might  be  carried  by  air  currents  and  infect  through  the 
respiratory  tract. 

The  fact  that  with  the  extermination  of  the  anopheles  varieties  mala- 
rial fevers  in  man  would  be  made  impossible  remains  established;  the 
parasite  must  have  its  chance  of  rejuvenescence  in  the  mosquito's 
stomach. 

Hitherto  no  experiments  have  been  made  to  show  the  possibility  of 
sporozoites  penetrating  the  capillary  walls  of  the  lung  tissue  or  those 
of  the  mucous  membranes. 

Malarial-like  Organisms  in  Other  Animals. — Two  varieties  of  malarial 
parasites  have  been  found  in  birds,  the  proteosoma  (Hcemamoeba 
relicta,  Cytasporon  danilewskyi)  and  the  Halteridium  (hsemoproteus). 
The  development  of  each  seems  to  be  very  similar  to  that  of  the  human 
malarial  organism.  The  life  cycle  of  the  proteosoma  is  the  better  known 
of  the  two.  The  asexual  cycle,  or  schizogony,  is  best  studied  in  artificially 
infected  birds  (canaries).  The  sexual  cycle  occurs  in  the  gnat  (Cidex 
pipiens). 

Conjugation  of  malarial  organisms  was  first  observed  by  MacCallum 
in  the  genus  Halteridium  in  birds,  and  his  discovery  gave  the  first  clue  to 
the  nature  of  the  flagella. 

-Malarial-like  organisms  have  also  been  found  in  monkeys,  cattle, 
and  frogs,  but  they  have  not  been  minutely  studied. 


33 


CHAPTEK  XLI. 

PIROPLASMA  BIGEMINUM— THE  MICROSPORIDIA— BALANTIDIUM 

COLL 

Genus  Piroplasma  Smith  and  Kilborne  (1893). 

IT  was  riot  until  1888  that  there  was  a  hint  as  to  the  real  nature  of  the 
actual  cause  of  "Texas  fever"  and  allied  diseases  which  attack  field 
cattle  in  many  parts  of  the  world.  Then  Babes  described  inclusions  in 
red  blood  cells  in  Roumanian  cattle  sick  with  the  disease,  though  he 
did  not  decide  upon  the  nature  of  the  organism.  No  new  studies  were 
reported  until  1893,  when  Theobald  Smith  and  Kilborne  gave  such  a 
complete  description  of  this  disease  and  its  cause  as  occurring  in  Texas 
cattle  that  little  that  is  new  has  since  been  discovered. 

These  authors  describe  as  the  cause  of  Texas  fever  pigment-free, 
amoeboid  parasites  appearing  in  various  forms  within  the  red  blood 

cells  of  infected  animals.  The  organ- 
isms may  be  irregularly  round  and  lie 
singly  or  they  may  be  in  pear-shaped 
twos,  united  by  a  fine  line  of  protoplasm. 
Because  of  these  double  pear-shaped 

Piroplasma  bigeminum  showing  pear-       forms    gmith    Rnd    Kilborne    named    the 
shaped   forms  in   curved  and  straight  .  , 

axes.  (After  Kossei  and  weber.)  organism    pyrosoma     bigemmum1    and 

placed    it    among     the    haemosporidia. 

These  authors  also  showed  that  the  contagion  was  carried  by  a  tick 
(Boophilus  bovis).  Their  work  has  been  corroborated  by  many  investi- 
gators in  different  parts  of  the  world. 

Morphology  of  the  Parasite. — In  the  examination  of  fresh  blood  of 
sick  cattle  under  1000  diameters,  according  to  Smith  and  Kilborne, 
are  seen,  in  the  red  blood  cells,  double  pear-shaped  forms  and  single 
rounded  or  more  or  less  irregular  forms.  The  size  varies,  though  gen- 
erally it  is  the  same  among  the  bodies  in  the  same  red  blood  cell.  The 
average  size  is  2/J.  to  4/*  long  and  1^/e  to  2/*  wide.  The  pointed  ends  of 
the  double  form  are  in  apposition  and  generally  touch,  though  in 
unstained  specimens  a  connection  between  them  cannot  be  seen.  The 
axis  forms  either  a  straight  line  or  an  angle.  The  protoplasm  has  a 
pale,  non-granular  appearance,  and  is  sharply  separated  from  the  proto- 
plasm of  the  including  red  blood  cell.  The  small  forms  are  generally 
fully  homogeneous,  whereas  the  larger  ones  often  contain  in  the  rounded 
ends  a  large  rounded  body,  O.I//  to  0.2//  in  size,  which  is  very  glistening 

1  The  generic  name  Pyrosoma,  already  in  use  for  a  well-known  Ascidian  genus,  was  altered  to 
Piroplasma  by  Patton  in  1895. 


PIROPLASMA  BIGEMINUM  515 

and  takes  a  darker  stain.  \Vitliin  the  largest  forms  in  the  centre  of  the 
thick  end  is  a  large  round  or  oval  body,  0.5/*  to  I//,  which  sometimes 
shows  amo'boid  motions.  The  motion  of  the  whole  parasite  on  the  warm 
stage  is  not  produced  by  the  formation  of  distinct  pseudopods,  but  by  a 
constant  change  of  the  boundary.  The  changes  can  succeed  each  other 
so  quickly  that  it  is  scarcely  possible  to  follow  them  with  the  eye.  The 
motion  may  persist  for  hours.  The  single  ones  show  motion,  while  the 
double  ones  remain  unchanged.  fThe  parasites  take  most  basic  aniline 
stains  well.  Methylene  blue  is  especially  recommended.  The  Roman- 
owsky method  or  its  modifications  gives  the  best  results. 

The  number  of  red  cells  infected  is  about  1  per  cent,  of  the  whole. 
If  the  number  increases  to  5  per  cent,  or  10  per  cent,  it  generally  means 
the  death  of  the  animal.  The  parasites  quickly  disappear  from  the 
blood  after  the  disappearance  of  the  fever.  In  fatal  cases  many  para- 
sites are  found  in  the  red  blood  cells  of  the  internal  organs.  They 
vary  in  number  according  to  the  stage  at  which  death  occurs,  are  most 
abundant  in  the  kidneys,  and  are  found  in  fewer  numbers  in  the  liver, 
spleen,  and  other  internal  organs. 

The  complete  life  cycle  of  the  organism  is  not  known.  According 
to  Smith  and  Kilborne  tiny  motile  spore  forms  enter  the  red  blood  cells 
and  divide,  the  two  parts  remaining  together  forming  diplococcus-like 
bodies.  These  forms  increase  in  size  and  produce  the  pear-shaped 
bodies.  How  the  small  forms  are  produced  from  these  is  not  known. 

R.  Koch  has  described  a  bacillar  form  which  he  found  in  large  num- 
bers in  red  blood  cells  of  acute  fatal  cases  in'  East  Africa.  Between 
these  and  the  pear-shaped  forms  he  found  all  grades.  He  considers 
them  young  forms. 

Ziemann  showed  by  the  Romanowsky  method  that  the  parasite  con- 
tained chromatin  staining  material  situated  at  or  near  its  periphery. 

Kossel  and  Weber,  who  studied  the  parasite  found  in  haemoglobinuria 
of  cattle  in  Finland,  give  the  following  description  of  the  specimens 
stained  according  to  Romanowsky: 

"  The  smallest  forms  appear  as  tiny  rings,  about  one-sixth  the  diameter 
of  the  red  blood  cell.  The  rim  of  the  ring  takes  the  red  stain,  while  the 
rest  appears  blue.  Forms  a  little  larger  are  irregular  in  outline  and 
already  show  an  arrangement  of  the  chromatin  into  two  parts,  which 
are  more  distinct  in  larger  parasites  and  which  finally  become  separated 
into  four  parts.  In  the  large,  double,  pear-shaped  parasites  the  chro- 
matin is  generally  situated  at  the  poles,  more  seldom  near  the  middle." 

No  division  forms  similar  to  thos?  seen  in  the  sporulating stage  or  schiz- 
ogonv  in  malaria  have  been  seen.  Neither  has  a  sexual  cycle  in  another 
host  similar  to  thatof  the  malarial  parasite  in  the  mosquito  been  observed. 
Smith  and  Kilborne  showed  that  the  infection  is  caused  by  the  larva  of 
a  species  of  tick,  Boophilus  bovis  Curtis  (rhipicephalus  annulatus),  and 
Kossel  gives  Ixodes  redivius  as  the  tick  causing  transmission  of  the  germ 
in  the  hremoglobiniiria  of  Finland  cattle.  We  know  nothing  of  any 
changes  going  on  in  the  parasite  in  its  passage  through  the  tick.  The 
ticks  feeding  upon  the  blood  of  cattle  and  other  mammals  become 


516  PROTOZOA 

sexually  mature  at  their  last  moult.  They  then  pair,  and  the  fertilized 
females,  after  gorging  themselves  with  the  blood  of  their  host,  drop  to 
the  ground.  Each  female  then  lays  about  2000  eggs,  and  within  the 
shell  of  each  egg  a  large  quantity  of  blood  is  deposited  to  serve  as  food 
for  the  developing  embryo.  The  female  then  shrivels  up,  becoming  a 
lifeless  skin.  The  newly  hatched  larvse  containing  in  their  abdomens 
some  of  the  mother-blood,  crawl  about  until  they  either  die  from  starva- 
tion or  have  the  opportunity  of  passing  to  the  skin  of  a  fresh  host.  If 
the  mother-tick  has  drawn  its  supply  of  blood  from  cattle  infected  with 
piroplasma,  her  larvae  are  born  infected  with  the  parasite  and  become 
the  means  of  disseminating  the  disease  further.  This  mode  of  dissemi- 
nation explains  the  long  incubation  period  of  the  disease  (forty-five  to 
sixty  days — thirty  days  for  the  development  of  the  larvaB  and  the  re- 
mainder for  the  development  of  the  parasite  within  the  host).  It  is  pos- 
sible that  the  tick  embryo  acquires  the  infection  secondarily  from  the 
blood  it  absorbs  in  the  egg,  and  that  the  parasites  do  not  pass  through 
the  ovum  itself  as  in  nosema  bombycis. 

It  is  not  known  whether  among  the  piroplasmata  described  as  occur- 
ring in  cattle  in  various  parts  of  the  world  there  are  different  varieties. 
They  seem  to  be  morphologically  very  similar  and  to  produce  similar 
diseases.  So  far  it  has  not  been  possible  experimentally  to  inoculate 
animals  other  than  cattle  with  these  parasites.  Calves  withstand  the 
infection  better  than  older  animals  and  a  certain  degree  of  immunity 
is  reached  in  some  of  the  older  cattle  in  infected  districts.  The  piro- 
plasmata taken  in  by  such  animals  may  remain  as  harmless  parasites 
for  some  time.  If,  however,  such  cattle  are  weakened  from  any  cause, 
their  resistance  to  the  organism  may  be  lowered  and  they  may  there- 
fore pass  through  a  more  or  less  severe  attack  of  the  disease. 

Symptoms  of  the  Disease. — Fever  (40°  to  42°  C.),  anorexia,  weakness, 
increased  pulse  and  respiration,  decreased  secretion  of  milk,  hsemo- 
globinuria  at  the  height  of  the  fever,  causing  the  urine  to  appear  dark 
red  like  port  wine  or  darker.  The  urine  may  contain  albumin  even  if 
the  hrcmoglobinuria  is  absent,  but  there  are  no  red  blood  cells  present, 
the  color  being  due  to  the  coloring  matter  only.  There  is  icterus  of  the 
mucous  membrane  if  much  blood  is  destroyed. 

The  prognosis  varies  in  different  epidemics  from  20  to  60  per  cent. 
Death  may  occur  in  three  to  five  days  after  first  symptoms  appear. 
Recovery  is  indicated  by  a  gradual  fall  of  the  fever. 

The  only  treatment  from  which  any  results  have  been  obtained  is 
quinine  in  large  doses.  This  seems  to  have  helped  in  some  epidemics. 

Prophylaxis. — Stalled  cattle  are  not  infected,  but  it  is  impracticable 
to  keep  large  herds  of  cattle  stalled.  If  the  cattle  are  kept  from  infected 
fields  for  one  or  two  years  and  other  animals  (horses  and  mules)  are 
allowed  to  feed  there  the  ticks  may  disappear.  The  burning  of  the 
field  for  one  season  may  have  a  good  effect.  If  animals  cannot  be  taken 
from  infected  fields  such  fields  should  be  enclosed. 

Ticks  on  animals  may  be  killed  by  allowing  the  cattle  to  pass  through 
an  oil  bath  (paraffin,  cottonseed  oil,  etc.),  whereupon  the  ticks  die  from 


PIROPLASMA  BIGEMINUM  517 

suffocation.  The  bath  should  be  repeated  after  a  week  in  order  to  kill 
any  lame  which  may  have  developed.  All  animals  sent  from  infected 
regions  should  receive  this  treatment.  Animals  apparently  healthy 
before  the  treatment,  after  the  disturbing  influence  of  the  bath  often 
develop  the  disease  in  an  acute  form  and  die. 

Certain  birds  in  Australia  seem  to  feed  on  the  ticks,  therefore  such 
birds  might  be  propagated. 

Various  attempts  have  been  made  to  give  protection  by  the  inoculation 
of  fresh  (not  older  than  two  to  three  days)  blood  from  slightly  infected 
animals.  Some  partial  results  have  been  reported,  especially  when  the 
inoculations  were  made  during  the  cold  months.  In  Australia  the 
inoculation  of  defibrinated  blood  from  animals  which  have  just  recov- 
ered from  the  infection,  but  whose  blood  still  contains  some  parasite, 
has  been  tried.  Such  inoculations  should  be  followed  by  a  slight  attack 
of  the  disease  in  order  to  give  protection.  So  far  no  absolute  protec- 
tion has  been  produced,  neither  does  the  parasite-free  serum  of  animals 
which  have  entirely  recovered  from  the  disease  seem  to  contain  pro- 
tective qualities. 

Haemosporidia  similar  to  those  described  in  the  hsemoglobinuria  of 
cattle  have  been  found  in  dogs,  sheep,  horses,  and  pigeons.  Nocard 
and  Motas,  who  have  made  the  most  complete  study  of  these  para- 
sites in  the  malignant  jaundice  or  hsemoglobinuria  of  dogs,  state  that 
though  the  parasites  are  morphologically  similar  to  those  infecting  cattle, 
yet  it  is  impossible  to  infect  cattle  or  any  other  animal  tried  with  them. 
They  form  therefore  a  physiological  variety. 

Piropiasmata  in  Human  Beings. — Recently  Wilson  and  Chowning  have 
reported  the  infection  of  man  by  an  organism  similar  to  the  piroplasma 
bigeminum.  The  cases  in  which  they  state  that  they  found  the  organism 
were  those  of  "spotted  fever"  ("black  fever,"  "blue  disease")  in  Mon- 
tana. According  to  these  investigators  the  first  case  of  this  fever  occurred 
in  this  vicinity  in  1873.  Since  then  probably  200  cases  of  the  severe  type 
have  occurred,  with  a  mortality  of  70  to  80  per  cent.  The  disease  occurs 
chiefly  in  the  spring.  The  symptoms  are  chill;  pains  in  joints;  consti- 
pation; fever  with  morning  remissions  reaching  103°  to  104°  on  the 
second  day  of  appearance  and  a  maximum  of  105°  to  107°  in  five  to 
seven  days,  diminishing  at  the  end  of  the  second  week,  and  normal 
two  to  four  weeks  later;  purpuric  eruption  over  the  entire  body,  appear- 
ing from  the  second  to  the  fifth  day  after  the  chill  and  reaching  a  maxi- 
mum in  one  to  two  days;  slight  jaundice;  muttering  delirium  just  before 
death;  pulse  and  respiration  very  rapid;  urine  slightly  albuminous, 
with  granular  and  blood  casts.  The  authors  studied  23  cases  during 
1902  and  1903,  and  in  all  of  these  they  say  that  they  found  the  organism, 
within  the  red  blood  cells  in  most  instances,  but  sometimes  between  the 
cells,  few  in  number  in  some  of  the  cases,  many  in  the  others.  The 
time  of  the  appearance  of  the  organism  during  the  course  of  the  disease 
has  not  been  determined.  The  authors  describe  the  organism  as  vary- 
ing somewhat  in  size,  form,  and  staining  reaction.  In  general,  they 
state  it  closely  resembles  the  piroplasma  bigeminum. 


518 


PROTOZOA 


Inoculation  experiments,  according  to  these  authors,  show  rabbits  to 
be  slightly  susceptible,  inasmuch  as  the  organisms  cause  slight  fever 
and  remain  for  a  long  time  in  the  blood  without  apparent  harm. 

The  mode  of  infection,  the  authors  think,  is  probably  through  the 
bite  of  ticks,  members  of  the  genus  Dermacentor.  These  results  have 
not  yet  been  corroborated.  On  the  contrary,  some  good  authorities 
state  that  the  bodies  described  by  Wilson  and  Chowning  as  organisms 
are  artifacts,  and  that  there  is  no  evidence  to  show  that  the  disease  in 
question  is  caused  by  a  protozoan. 

Cnidosporidia. 

Subclass:  Neosporidia. 

Order:  Cnidosporidia. 

Suborder:  Microsporidia. 

The  Cnidosporidia,  or,  as  the  whole  group  is  still  sometimes  called, 
the  myxosporidia,  is  one  of  the  most  populous  and  abundant  groups 
of  the  sporozoa,  showing  great  structural  variation  as  well  as  divergence 
in  mode  of  life.  Nevertheless  the  members  have  as  a  group  the  follow- 


FlG.  158 


Nosema  bombycis :  1  to  5,  spore  formation ;  6,  infected  follicle  of  testicle ;  7,  spores ;  a,  b,  fresh ; 
c,  d,  treated  with  nitric  acid.  The  acid  causes  them  to  swell  up  and  increase  in  size  by  at  least  a 
half,  at  the  same  time  making  the  polar  capsule  distinct.  In  d  the  filament  is  extruded.  (After 
Balbiani.) 

ing  well-marked  characteristics:  The  trophozoite  is  amoeboid;  spore 
formation  begins  at  an  early  period  and  proceeds  continuously  during 
the  growth  of  the  trophozoite;  the  spores  are  produced  endogenously — 
i.  e.,  within  the  protoplasm  of  the  trophozoite,  and  each  spore  always 
possesses  one  or  more  very  distinctive  structures,  "the  polar  capsules." 
The  Cnidosporidia  are  habitants  of  fishes,  reptiles,  arthropods,  and 
some  other  classes  of  animals. 

The  microsporidia  infest  especially  arthropods,  causing  often  most 
virulent  epidemics.     The  most  interesting  member  of  this  group  is 


BALANTIDIUM 


519 


Nosema  bombycis,  the  cause  of  silkworm  disease  (Pe*brine).  The 
organism  forms  many  small  spores  each  with  one  polar  capsule.  The 
spores  a iv  carried  1>\  the  food  into  the  intestinal  canal  of  the  caterpillar, 
pass  through  the  walls  of  the  intestines,  and  infect  all  organs.  Spores 
found  in  the  ovary  may  be  carried  over  to  the  newly  hatched  silkworms, 
thus  causing  a  further  dissemination  of  the  disease. 

The  other  member  of  this  group  of  interest  here  is  Nosema  lophii 
(I)oHein).  Its  interest  lies  in  the  fact  that  it  has  been  found  to  infect  only 
the  ganglion  cells  of  the  sea-devil,  thus  apparently  resembling  in  its  para- 
sitic1 nature  the  organism  causing  hydrophobia. 

Heterotricha. 

Class:  Cilia ta. 

Order:  Heterotricha. 

Genus:  Balantidium. 

Balantidium  coli  (Malmst,  1857) .  The  body  of  this  infusorium  is  egg- 
shaped,  with  a  funnel-shaped  mouth  opening.  The  surface  of  the  body 
is  covered  with  a  pellicula,  under  which  is  a  distinct  ectoplasmatic  sheath 
containing  rows  of  basal  granules  from  which  the  short,  fine  cilia  arise. 


FIG.  159 


12  3 

Balantidium  coli :  1,  2,  stages  of  division  ;  3,  conjugation.    (After  Leuckart.) 

The  cloudy  entoplasm  contains  fat  and  starch  granules  and  may 
contain  many  red  blood  cells  and  other  food  particles  from  the  host. 
Two  contractile  vacuoles  have  been  seen.  Posteriorly  there  is  a  small 
prominence  marking  the  place  where  excreta  are  expelled.  The  chro- 
matic macroniK'leus  is  bean-shaped,  and  the  vesicular  micronucleus 
is  nearly  spherical. 

Division  is  transverse,  the  macronucleus  dividing  by  simple  con- 
striction and  the  micronucleus  by  mitosis.  Conjugation  has  been 
observed.  Spherical  cysts  surrounded  by  a  thick  membrane  are  formed. 

The  balantidium  coli  has  been  found  in  the  large  intestine  of  human 
beings  and  of  swine — probably  two  distinct  varieties.  The  variety 


520  PROTOZOA 

occurring  in  human  beings  has  been  found  in  about  60  cases,  principally 
in  Sweden,  but  also  in  Russia,  Scandinavia,  Finland,  China,  Italy,  Ger- 
many, and  the  United  States.  Most  of  these  cases  were  suffering  from 
severe  chronic  intestinal  catarrh,  often  accompanied  by  bloody  diar- 
rhrea.  A  number  of  observers  think  the  balantidium  the  primary 
cause  of  the  catarrh,  others  think  it  a  secondary  excitant,  while  still 
others  believe  it  to  be  a  harmless  inhabitant  of  the  intestines. 

Schaudinn  has  described  two  additional  species  of  balantidium  found 
in  the  human  intestines,  which  he  has  called  Balantidium  minutum  and 
Nyctootherus  faba,  probably  both  non-pathogenic. 


CHAPTER   XLII. 

PROTOZOAN-LIKE  BODIES  IN  SMALLPOX  AND  ALLIED  DISEASES 
(COWPOX,  HORSEPOX,  SHEEPPOX)  AND  IN  SCARLET  FEVER. 

THE  question  as  to  the  chief  exciting  factor  in  smallpox  and  allied 
diseases,  according  to  some  authorities,  is  still  undecided,  while  accord- 
ing to  others  it  is  settled  beyond  doubt.  These  latter  investigators  con- 
sider that  certain  bodies  found  chiefly  in  the  epithelial  cells  of  the  skin 
and  mucous  membranes  in  the  specific  lesions  of  these  diseases  are 
protozoa  causing  the  diseases. 

The  different  diseases  named  in  the  chapter  heading,  excepting  only 
scarlet  fever,  if  not  identical,  are  closely  allied.  Indeed,  the  following 
facts  seem  to  prove  that  at  least  cowpox  and  variola  are  very  closely 
related  diseases,  if  not  the  same  disease :  First,  smallpox  virus  inocu- 
lated into  calves  produces  after  passage  through  several  animals  an 
affection  exactly  similar  to  cowpox.  The  successful  inoculation  of  the 
first  series  of  cattle  from  smallpox  is  a  matter  of  great  difficulty,  but  so 
many  experimenters  have  asserted  that  this  has  been  done  that  there 
seems  to  be  no  doubt  as  to  its  truth.  In  our  laboratory  not  one  of  many 
attempts  to  accomplish  it  has  been  successful.  Second,  both  when  occur- 
ring in  nature  and  when  produced  by  experiment  the  lesions  of  the  two 
diseases  are  similar.  Third,  monkeys  have  been  successfully  protected 
against  either  disease  by  previous  inoculation  of  the  other;  also,  obser- 
vations go  to  show  that  human  beings  inoculated  with  cowpox  vaccine 
are  not  susceptible  to  inoculation  with  smallpox  virus,  and  that  those 
who  have  within  a  varied  time  passed  through  an  attack  of  smallpox 
cannot  be  inoculated  successfully  with  cowpox  vaccine.  These  facts 
seem  positively  to  prove  that  the  two  diseases  are  produced  by  organ- 
isms originally  identical,  one  being  modified  by  its  transmission  through 
cattle,  the  other  through  human  beings. 

The  immunity  caused  by  successful  vaccination  is  not  permanent,  and 
varies  in  its  duration  in  different  individuals.  Although  it  may  give  some 
protection  from  smallpox  for  ten  or  fifteen  years,  it  is  not  well  to  count 
on  immunity  for  more  than  two  years,  and  whenever  one  is  liable  to 
exposure  it  is  well  to  be  vaccinated.  If  this  vaccination  were  unnecessary 
it  will  not  be  successful,  while  if  it  is  successful  we  have  reason  to 
believe  the  individual  was  open  to  at  least  a  mild  smallpox  infection. 

Protective  Substances  Present  in  the  Serum  of  Animals  after  Successful 
Vaccination. — It  has  been  frequently  shown  that  the  blood  serum  of  a 
calf  for  several  weeks  after  successful  vaccination  possesses  feeble 
protective  properties,  so  that  the  injection  of  one  to  two  litres  of  it  into 
a  susceptible  calf  would  prevent  a  successful  vaccination.  A  further 


522  PROTOZOA 

and  more  convincing  fact  has  been  demonstrated  by  Huddleston 
and  others,  namely,  that  when  active  vaccine  is  mixed  in  certain  pro- 
portions with  serum  from  an  animal  which  had  just  recovered  from  a 
successful  vaccination,  and  the  mixture  is  inoculated  into  a  susceptible 
animal,  there  is  no  reaction. 

Etiology  of  Variola  and  Cowpox. — It  has  been  repeatedly  shown  that 
no  bacteria  similar  to  any  of  the  known  forms  have  a  causal  relation 
to  these  diseases.  In  our  own  laboratory  we  are  able,  by  the  inoculation 
of  rabbits'  skins,  to  produce  extremely  active  vaccine  virus  in  large 
quantities,  absolutely  free  from  micro-organisms  as  grow  under  the  con- 
ditions of  our  present  methods  of  bacterial  cultivation.  Such  pure  active 
vaccine,  when  emulsified  in  equal  parts  of  glycerin  and  water  and  fil- 
tered through  two  or  three  thicknesses  of  the  finest  filter  paper,  gives  a 
slightly  opalescent  filtrate,  which  in  the  hanging  drop  under  high  magni- 
fication shows  many  very  tiny  granules  with  an  occasional  larger  one, 
and  in  smears  shows  no  formed  elements  giving  characteristic  stains. 
This  filtrate,  from  which  no  growth  can  be  obtained  on  artificial  culture 
media,  when  rubbed  over  a  freshly  shaved  rabbit's  skin,  after  the 
method  of  Calmette  and  Gue*rin,  gives  an  abundant  typical  reaction. 

These  facts  show  that  some,  at  least,  of  the  infective  forms  cannot  as 
yet  be  made  to  grow  outside  of  the  body,  that  such  forms  are  very  tiny, 
and  that  they  do  not  stain  characteristically  with  our  usual  methods  of 
staining.  We  have  shown  also  that  the  infective  agent  cannot  pass  an 
ordinary  Berkefeld  filter  under  forty  pounds'  pressure,  which  practi- 
cally rules  out  ultramicroscopic  forms. 

Since  Guarniere  in  1892  claimed  that  certain  inclusions  present  in 
the  epithelial  cells  of  the  lesions  of  smallpox  in  a  rabbit's  cornea  (Fig. 
160)  were  parasites,  much  attention  has  been  given  to  the  study  of  these 
bodies,  commonly  known  as  "vaccine  bodies,"  yet  opinions  still  differ  as 
to  their  nature.  The  most  recent  studies  of  importance  of  these  bodies 
have  been  made,  on  the  one  hand,  by  Councilman  and  his  associates,  who 
believe  them  to  be  protozoa,  and,  on  the  other,  by  Ewing,  who  believes 
that  all  of  the  forms  so  far  described  are  degeneration  products,  some 
specific,  others  not. 

Councilman  believes  that  there  are  two  cycles  of  development  of  the 
"parasite,"  one  intracellular  and  the  other  intranuclear,  arid  that  the 
intranuclear  infection  occurs  only  in  smallpox.  The  intracellular  cycle 
is  simple,  showing  only  "multiplicative  reproduction,"  while  the  intra- 
nuclear cycle  is  more  complicated,  probably  sexual  in  character. 
Calkins,  working  with  Councilman,  has  described  an  elaborate  cycle 
of  development  in  which  we  believe  are  included  many  forms  due  to 
degeneration  of  the  host  cells  alone. 

In  our  own  work  on  sections,  which  has  extended  irregularly  over  a 
period  of  several  years,  we  have  gotten  results  which  are  somewhat  con- 
fusing, principally  so  because  of  the  non-uniformity  of  the  appearances 
of  these  bodies,  both  by  different  methods  and  by  the  same  methods  at 
different  times.  There  is  no  doubt  that  whatever  the  nature  of  the  bodies 
they  are  easily  affected  by  methods  used  for  fixing,  hardening,  and  staining 


PROTOZOAN -LIKE  BODIES  IN  SMALLPOX 


523 


them.  This  accounts  in  part  for  the  varied  results  reported.  However,  in 
the  most  perfectly  prepared  specimens,  judged  according  to  the  appear- 
ance of  the  red  blood  cells,  leukocytes,  and  tissue  cells  at  a  distance 
from  the  lesions,  we  have  found  the  vaccine  bodies,  especially  in  corneal 

FIG.  160 


Epithelial  cells  of  a  rabbit's  cornea,  containing  "  vaccine  bodies."    Tissue  fixed  three  days  after  inocu- 
lation with  smallpox  virus,    a  and  d,  vaccine  bodies ;  b  and  c,  nuclei.    X  1500  diameters. 

infection,  to  show  a  more  or  less  constant  series  of  changes,  somewhat 
similar  to  those  described  by  Calkins  in  his  "gemmule  formation" 
and  by  Tyzzer  in  his  development  of  the  vaccine  bodies.  This  series 
of  changes  might  be  represented  somewhat  schematically  in  Fig.  161. 

FIG.  161. 


Schematic  representation  of  vaccine  bodies  seen  within  the  epithelial  cells  in  the  lesions  of  smallpox 
and  vaccinia :  1,  spore  (merozoite,  sporozoite  ?) ;  2,  small  form  which  stains  solidly  with  basic  stains ; 
3,  larger  form  which  contains  central,  more  darkly  staining  granule ;  4,  larger  form,  with  more  lightly 
staining  reticular  cytoplasm.  This  form  and  the  next  may  have  amoeboid  outline,  and  there  may  be 
larger  am<eboid  forms  which  might  be  interpreted  either  as  the  grown  single  form  or  as  the  fusion  of 
two  or  more  forms ;  5,  form  containing  two  central,  darkly  staining  bodies ;  6,  form  containing  many 
bodies  taking  basic  stains  more  or  less  intensely;  7,  form  containing  a  central  body  staining  faintly 
with  basic  dyes,  and  small  rounded  bodies  about  it,  some  taking  basic  and  some  acid  stains ;  8,  same 
as  7,  except  that  many  of  the  bodies  surrounding  the  central  body  are  definitely  ring-shaped,  and  all 
take  the  acid  stain.  These  forms  vary  in  size  ;  some  are  larger  than  the  host  nucJeus ;  9,  form  break- 
ing up  (spores  set  free?). 

( )ne  can  easily  see  that  such  tiny  bodies  as  these  possible  spores  with 
no  definite  characteristic  Maining  (jualities  would  be  with  difficulty, 
if  at  all,  differentiated  from  the  mass  of  cell  granules  in  the  degenerated 
areas  of  the  lesion,  and,  as  the  outline  and  structure  of  most  of  the  other 
forms  seem  to  be  easily  disturbed,  the  whole  question  as  to  their  nature 
is,  from  a  morphological  standpoint  alone,  a  very  diffcult  one  to  settle. 


524  PROTOZOA 

Our  best  results  on  corneas  have  been  obtained  with  the  following 
technique :  Fix  in  Zenker's  fluid  for  from  four  to  eight  hours ;  wash  in 
running  water  over  night;  place  in  95  per  cent,  alcohol  (changing  in 
two  hours  to  fresh)  for  twenty-four  hours,  then  in  absolute  alcohol  for 
twenty-four  hours.  Imbed  in  paraffin.  The  cuts  should  be  from  3/>«  to 
5,«  thick.  Stain  with  (1)  eosin  and  methylene  blue — eosin  half  an  hour, 
methylene  blue  two  minutes;  (2)  Heidenhain's  iron  hsematoxylin ;  (3) 
Bovrel,  modified  by  Calkins. 

Susceptibility  of  Different  Animals. — Horses,  rabbits,  sheep,  monkeys, 
and  guinea-pigs  are  susceptible.  The  pulp  and  serum  obtained  from 
an  epidemic  of  cowpox  took  feebly  in  calves  in  a  moderate  percentage 
of  those  inoculated.  The  characteristic  vaccine  bodies  were  found 
practically  identical  with  those  in  vaccinia,  except  that  the  bodies  were 
a  little  larger  and  more  irregular  in  outline. 

The  Preparation  of  Vaccine. — For  the  following  suggestions  I  am 
indebted  to  Dr.  J.  H.  Huddleston,  who  has  had  the  immediate  charge 
of  the  production  of  vaccine  for  the  New  York  Health  Department 
for  some  years: 

SEED  VIRUS. — A  sufficient  amount  of  vaccine  virus  should  be  on 
hand  to  vaccinate  forty  to  fifty  persons.  Five  children  in  good  health, 
and  not  previously  vaccinated,  should  then  be  vaccinated  each  in  a  spot 
the  size  of  a  ten-cent  piece.  On  the  fifth  day  after  vaccination  the  top 
of  the  resulting  vesicle  should  be  removed  and  sterilized  bone  slips  be 
rubbed  on  the  base  thus  exposed.  It  should  be  possible  in  this  manner 
to  charge  at  least  from  one  to  two  hundred  slips  on  each  side  of  the  slip 
from  each  child.  The  slips  should  be  allowed  a  moment  in  which  to 
dry  and  then  placed  in  a  sterilized  box,  in  which,  if  kept  in  cold  storage, 
they  will  probably  remain  efficient  for  at  least  two  or  three  weeks. 

ANIMALS. — The  preferable  animals  are  female  calves,  from  two  to 
four  months  of  age,  in  good  condition  and  free  from  any  skin  disease. 
These  can  easily  be  vaccinated  on  the  posterior  abdomen  and  inside 
of  the  thighs  by  placing  them  on  an  appropriate  table.  It  is  possible 
that  on  account  of  the  character  of  the  available  supply  older  animals 
may  be  desirable,  but  the  calves  take  more  typically  and  are  more 
easily  handled.  When  an  animal  is  too  old  to  be  thrown  and  held 
without  difficulty  it  may  be  vaccinated  on  the  rump,  each  side  of  the 
spine;  but  the  skin  there  is  tougher  than  on  the  posterior  abdomen 
and  inside  of  the  thighs,  and  the  resulting  virus,  though  efficient,  is 
not  so  easily  emulsified. 

VACCINATION. — The  calf  should  be  cleaned  thoroughly,  including 
the  feet  and  the  tail,  and  the  hair  should  be  clipped  from  the  end  of  the 
tail.  Next,  the  posterior  abdomen  and  insides  of  the  thighs  are  shaved 
and  the  skin  beneath  washed  in  succession  with  soap  and  water,  ster- 
ilized water  and  alcohol,  and  then  dried  with  a  sterile  towel.  On  this 
area  there  are  now  made  about  one  hundred  scarifications,  each  from 
one-quarter  to  one-half  of  an  inch  square.  The  scarification  is  made 
most  easily  by  cross-hatching  with  a  six-bladed  instrument,  the  blades 
being  about  one-thirtieth  of  an  inch  apart.  The  scarification  is  super- 


PROTOZOAN-LIKE  BODIES  IX  SMALLPOX  525 

ficial,  but  brings  a  small  amount  of  blood.  An  area  as  small  as  speci- 
fied is  less  likely  to  become  infected  than  a  larger  one.  The  scarifica- 
tions should  be  separated  from  each  other  by  an  interval  of  at  least 
one-half  to  three-quarters  of  an  inch.  After  they  have  been  made 
they  should  be.  dried  with  a  sterile  towel  or  with  sterile  cotton  and 
rubbed  with  the  charged  slips.  One  to  two  slips,  depending  on  the 
amount  of  virus  each  slip  holds,  should  be  sufficient  for  vaccinating 
each  vesicle. 

COLLECTION. — On  the  fifth  or  sixth  day,  depending  upon  the  rate 
of  development  of  the  vaccine  vesicles,  they  should  be  ready  for  col- 
lection. The  entire  shaved  area  is  washed  with  sterile  water  and  sterile 
cotton,  and  the  crusts  are  picked  off.  The  soft,  pulpy  mass  remaining 
is  then  curetted  off  with  an  ordinary  steel  curette  and  the  pulp  placed 
in  a  sterilized  vessel.  After  the  curettage,  serum  exudes  from  the  torn 
base  of  the  vesicle,  and  ivory  slips  may  be  charged  in  this.  The  pulp 
should  be  mixed  with  from  two  to  three  times  its  weight  of  glycerin 
and  water,  equal  parts,  and  this  is  done  most  effectively  by  passing  the 
mixture  between  the  rollers  of  a  Doring  mill.  The  more  watery  the  pulp, 
especially  if  it  is  not  to  be  used  immediately,  the  smaller  should  be  the 
proportion  of  glycerin.  The  emulsion  so  produced  can  then  be  put  up 
for  issue  in  vials.  The  slips  charged  with  the  serum  from  the  calf  may 
also  be  used  for  vaccinating.  Capillary  tubes  require  especial  means  of 
filling,  and  small  vials  filled  and  corked  answer  the  purpose  admirably. 

PROPAGATION. — Subsequent  animals  may  be  vaccinated  in  any  one 
of  the  three  ways:  (a)  slips  may  be  charged  from  typical  vesicles  on 
primary  vaccinations,  just  as  with  the  first  calf,  and  used  for  seed  virus; 
(6)  slips  charged  with  the  serum  from  the  calf  may  be  used  to  vaccinate 
a  second  calf;  (c)  the  glycerinated  emulsion  may  be  used  to  vaccinate 
succeeding  calves,  but  in  the  last  case  it  is  necessary  to  keep  the  emulsion 
a  varying  length  of  time — often  two  or  three  months — before  it  is  fit 
for  use  in  vaccination  of  the  calf,  since  the  employment  of  fresh 
glycerinated  pulp  on  a  succession  of  calves  leads  to  prompt  degenera- 
tion of  the  vaccine  and  to  the  production  of  infected  vesicles. 

Care  of  the  Calves.— All  bedding  is  avoided  and  an  exclusively  milk 
diet  given;  thus  much  of  the  otherwise  unavoidable  dust  is  done  away 
with. 

Laboratory. — The  laboratory  should  consist  of  at  least  three  rooms: 
(a)  stable;  (6)  operating-room;  (c)  laboratory-room.  It  should  be 
possible  to  make  and  keep  all  the  rooms  clean.  The  stable  and  oper- 
ating-room should  be  flushed  with  a  hose  and  hot  water  daily.  Excreta 
should  be  removed  immediately.  The  calves  can  be  kept  clean  if 
they  stand  on  a  raised  and  perforated  platform,  which  is  so  short  that 
the  defecations  cannot  fall  on  it,  and  if  they  have  no  bedding.  They 
must  be  fastened  to  keep  them  from  licking  the  scarifications.  In  the 
health  department,  when  a  calf  is  removed,  its  stall  and  platform  are 
scoured  with  a  brush  and  sodium  carbonate  solution.  The  stable 
should  he  provided  with  a  shovel,  broom,  hose,  currycomb,  mane  brush, 
cord,  and  with  halters,  buckets,  scrubbing  brushes,  and  sponges.  The 


526  PROTOZOA 

operating-room  should   be  well  lighted  and  provided  with  a  table  and 
with  stools. 

The  only  requisites  for  the  table  are  that  it  should  be  heavy  and  firm ; 
that  it  should  have  holes  through  the  top  so  arranged  that  straps  can 
be  passed  through  them  to  hold  the  calf  down,  and  a  vertical  strip  on 
one  side  of  the  table  to  which  the  upper  hind  leg  of  the  calf  can  be 
fastened.  The  calf  can  be  thrown  upon  the  table  easily  by  two  attend- 
ants. 

The  laboratory  should  also  be  well  lighted  and  furnished  with  tables, 
chairs,  desk,  case  for  instruments,  and  refrigerator.  It  should  also 
have  both  a  steam  and  a  dry-air  sterilizer,  a  set  of  scales  weighing  to 
grams  or  centigrams,  and  a  blast  lamp  and  bellows.  In  stock  there 
should  be  one  to  two  thousand  bone  slips  for  seed  virus,  and  ten  to 
fifteen  thousand  smaller  slips  for  issue;  two  or  more  scarifiers;  a  curette; 
four  to  six  razors  for  shaving  the  animals;  a  razor  strop;  a  pair  of  large 
scissors,  curved  on  the  flat,  for  clipping  the  animals;  a  burette,  from 
which  glycerin  flows  while  the  vaccine  pulp  is  being  ground;  burette 
holder;  a  Doring  vaccine  grinder;  clinical  thermometers  to  take  the 
temperature  of  the  animals;  six  to  twelve  small  glass  dishes  with  covers; 
a  hard-rubber  syringe,  of  four-ounce  capacity,  to  make  suction ;  absorb- 
ent cotton;  glass  vials  and  corks;  and  several  pounds  of  soft  glass 
tubing,  three-eighths  of  an  inch  in  calibre,  to  store  virus  emulsion. 
There  should  also  be  gowns  and  caps  for  the  attendants.  Sodium 
carbonate,  bichloride  of  mercury,  bromine  for  a  deodorizer,  alcohol, 
and  glycerin  are  the  chemicals  needed. 

For  issue  for  public  vaccinations  there  are  also  needed  packing- 
boxes,  rubber  bands,  sheet  wadding,  needles,  and  wooden  toothpicks 
for  removing  the  virus  from  the  vials  and  rubbing  it  on  the  scarifica- 
tions. 

Yield. — The  material  obtained  from  the  five  children  should  vaccinate 
at  least  five  calves;  it  may  easily  vaccinate  fifteen  calves.  Ten  grams 
of  pulp  and  two  hundred  charged  slips  would  be  an  average  yield  from 
a  calf,  and  that,  when  made  up,  should  suffice  to  vaccinate  at  least 
fifteen  hundred  persons.  Calves  vary  immensely  in  the  yield.  Of  two 
calves  vaccinated  in  precisely  the  same  way  one  may  furnish  material 
for  five  hundred  vaccinations  and  the  other  for  ten  thousand  vaccina- 
tions. 

The  Durability  of.  Glycerinated  Virus  in  Sealed  Tubes. — As  a  result  of 
testing  from  time  to  time  an  immense  number  of  specimens  of  vaccine, 
the  conclusion  has  been  reached  that  vaccine  properly  put  up  should 
keep  at  least  three  months.  From  time  to  time  a  single  lot  of  virus  will 
fail  by  the  end  of  one  month.  Sometimes  this  is  due  to  the  glycerin, 
as  when  it  has  some  chemical  impurity,  or  as  when  simply  it  is  not 
diluted  sufficiently  with  water.  We  find  that  one  part  of  water  to  two 
of  glycerin  makes  a  good  dilution. 

Bacteria  in  Vaccine. — It  is  impossible  to  prepare  vaccine  on  a  large 
scale  so  that  it  is  at  the  time  of  its  removal  free  from  bacteria.  In  fact, 
there  are  usually  very  large  numbers  of  one  or  more  varieties  of  bacteria 


PLATE   IV. 


gr-T^^r^       5  /-^ssr 

/•;! 


m 

•  ^ZLi 


• 


II 


III 


Protozoan-like  Bodies  in  Scarlet  Fever  (1-7)  and 
Measles  (I-TII). 


PROTOZOAN-LIKE  BODIES  IN  SCARLET  FEVER  527 

present.  When  the  stable  and  animals  have  been  kept  clean  the  bacteria 
comprise  usually  very  few  varieties;  when  dirty  conditions  prevail  the 
bacterial  varieties  are  more  numerous.  The  number  of  bacteria  found 
varies  enormously.  The  largest  number  found  by  us  in  vaccine  pulp 
from  the  calf  was  126,360  in  one  loopful,  and  the  smallest  number  523. 
Discrete  vesicles  at  the  borders  contain  many  less  bacteria  than  the  con- 
Huent  ones  caused  by  the  inoculation  at  the  scarification.  The  pulp 
has  many  more  bacteria  than  the  serum  of  the  vesicles.  The  period 
which  elapses  before  glycerinated  virus  becomes  sterile  is  also  quite 
variable,  but  does  not  depend  in  any  direct  way  upon  the  number  of  bac- 
teria originally  present.  A  very  large  number  may  disappear  rapidly, 
and  a  few  persist  for  a  long  time.  Upon  rabbits  a  practically  bacteria- 
free  vaccine  can  be. obtained. 

After  two  or  three  weeks  the  number  of  living  bacteria  is  usually 
greatly  diminished,  especially  after  addition  of  glycerin,  but  seldom 
is  absolutely  nil.  If  we  wait  until  the  vaccine  is  surely  sterile  it  is 
very  apt  to  be  also  useless — that  is,  by  that  time  the  specific  organisms, 
too,  as  well  as  the  bacteria,  are  dead. 

In  a  very  large  experience  we  have  learned  that  the  number  of  bac- 
teria present  has  little  to  do  with  the  result  of  the  vaccination. 

Pathogenic  bacteria  other  than  the  practically  non-virulent  skin 
staphylococci  are  not  found  when  animals  are  properly  kept  and  vac- 
cinated. The  vaccine  pulp  and  serum  mixture  is  added  to  two  and 
one-half  to  three  and  one-half  times  its  bulk  of  a  mixture  consisting  of 
two  parts  of  chemically  pure  glycerin  and  one  part  of  water. 

Efficient  vaccine  should  be  inoculated  in  a  portion  of  skin  no  more 
than  one-eighth  inch  in  diameter. 

Scarlet  Fever. 

Very  recently  Mallory  has  reported  the  presence  of  certain  bodies 
in  scarlet  fever.  He  summarized  his  observations  as  follows:  "In  4 
cases  of  scarlet  fever  certain  bodies  were  found  which  in  their  mor- 
phology strongly  suggest  that  they  may  be  various  stages  in  the  devel- 
opmental cycle  of  a  protozoan.  They  occur  in  and  between  the  epithe- 
lial cells  of  the  epidermis  and  free  in  the  superficial  lymph  vessels 
and  spaces  of  the  corium.  The  great  majority  of  the  bodies  vary  from 
2/Jt  to  7/i  in  diameter,  and  stain  delicately  but  sharply  with  methylene 
blue.  They  form  a  series  of  bodies,  including  the  formation  of  definite 


I)K><  KIPTION  OF  PLATE  IV. 

Photographs  of  three  forms  of  the  small  bodies  found  in  blister  fluid  from  cases  of  measles. 
I.  Small  form  with  central  chromatin  mass. 

II.  Medium-sized  form  with  chromatin  granules  distributed  throughout  its  protoplasm. 
III.  Large  form  with  most  of  the  chromatin  granules  peripherally  arranged. 
Protozoan-like  bodies  in  scarlet  fever.    Stained  with  eosin  and  methylene  blue.    1,  numerous  large 
and  small  scarlet  fever  bodies  (stained  light  blue)  in  and  between  the  epithelial  cells  of  the  rete 
mucosum.    Several  of  the  bodies  suggest  fixation  while  in  amoeboid  motion  ;  2,  coarsely  reticulated 
form  which  may  be  degenerated  form  or  stage  in  sporogony  ;  3,  probable  stage  preceding  the  radiate 
bodies ;  4,  5,  6,  and  7,  different  stages  in  the  development  of  the  radiate  bodies.    (After  Mallory.) 


528  PROTOZOA 

rosettes  with  numerous  segments,  which  are  closely  analogous  to  the 
series  seen  in  the  asexual  development  (schizogony)  of  the  malarial 
parasites,  but  in  addition  there  are  certain  coarsely  reticulated  forms 
which  may  represent  stages  in  sporogony  or  be  due  to  degeneration  of 
the  other  forms."  In  our  laboratory  nine  cases  have  been  examined 
with  reference  to  the  presence  of  these  bodies,  five  autopsy  cases  and 
four  living,  but  so  far  only  a  few  of  the  less  characteristic  forms  have 
been  found,  and  these  only  in  the  five  autopsy  cases. 

Duval  has  just  made  the  announcement  that  in  fluid  obtained  through 
blistering  the  skin  of  scarlet  fever  patients  by  a  very  quick  method  he 
has  obtained  bodies  which  he  interprets  as  forms  of  Mallory's  protozoan. 

In  our  laboratory  Field  has  obtained  similar  bodies  by  the  same 
method  in  both  scarlet  fever  and  measles  cases,  and  in  four  cases  of 
scarlatiniform  antitoxin  rashes,  more  in  the  first  two  groups  than  in  the 
last.  He  has  obtained  them  in  no  other  cases  so  far  examined.  Nothing 
positive  can  be  said  yet  as  to  the  nature  of  these  bodies,  though  Field 
has  come  to  the  conclusion  that  the  majority  of  them  are  from  degen- 
erated leukocytes. 


CHAPTER  XLIII. 

KALA-AZAR— RABIES. 
Eala-azar. 

CERTAIN  fevers  of  severe  malarial-like  types  known  in  different  sec- 
tions of  the  tropics  by  different  names  (dum-dum  fever,  cachexial 
malaria,  kala-azar)  have  recently  been  shown  to  be  due  to  the  same 
cause  by  the  finding  of  similar  protozoan-like  bodies  in  the  lesions. 
These  bodies  were  first  minutely  described  by  Leishman  in  1900  as 
being  present  in  certain  cells  in  the  spleen  of  cases  called  by  him  dum- 
dum fever  occurring  in  India.  In  1903  Donovan  described  similar 
bodies  in  cases  of  what  he  called  malarial  cachexia.  The  bodies  have 
been  called  the  Leishman-Donovan  bodies  or,  more  properly,  the  Leish- 
man bodies.  They  have  since  been  found  in  different  parts  of  India, 
in  China,  Tunis,  Algiers,  Arabia,  and  Egypt,  and  quite  recently  Wright 
has  reported  in  a  case  of  Delhi  boil  from  Armenia  bodies  which,  accord- 
ing to  his  excellent  photographs  and  description,  must  be  identical  with 
or  very  closely  related  to  Irishman's  bodies,  though  he  does  not  call 
attention  to  that  fact. 

The  bodies  have  been  found  in  large  endothelioid  cells  in  the  spleen, 
liver,  mesenteric  glands,  bone-marrow,  kidney,  lungs,  testes,  skin,  and 
ulcers  in  intestines. 

The  symptoms,  in  the  cases  of  general  infection  are:  (1)  very  much 
enlarged  spleen  and  less  enlarged  liver;  (2)  progressive  anaemia  with 
peculiar  earthy  pallor  of  skin,  progressive  emaciation,  and  muscular 
atrophy;  (3)  long-continued,  irregularly  intermittent  fever  (97°  to  104°) ; 
(4)  hemorrhages,  such  as  epistaxis,  bleeding  from  gums  into  sub- 
cutaneous tissue,  producing  purpuric  eruption;  (5)  transitory  oedemas 
of  various  regions.  There  are  often  complications  such  as  congestion 
of  lungs,  dysentery,  cancrum  oris.  The  blood  count  shows  practically 
no  loss  of  haemoglobin,  but  there  is  a  decrease  in  the  leukocytes,  prin- 
cipally polynuclears,  giving  a  relative  increase  of  mononuclears. 

Negative  points  which  help  in  the  diagnosis  are :  absence  of  malaria, 
no  typhoid  or  Malta  fever  reaction,  resistance  to  medication,  quinine, 
as  a  rule,  having  no  effect,  though  in  early  cases  a  few  good  results 
have  been  reported.  Splenic  puncture  with  the  finding  of  Leishman 
bodies  makes  the  diagnosis  certain.  The  duration  of  the  disease  is 
from  a  few  months  to  several  years.  The  percentage  of  deaths  is  great; 
in  some  forms  of  the  disease  it  may  reach  90  per  cent. 

Morphology. — The  bodies  are  circular  to  elliptical  in  shape,  from  2/J.  to 
4/JL  in  diameter,  and  contain  a  double  nucleus,  a  large  oval  one  at  one  part 
of  the  periphery  and  a  small  circular  or  rod-shaped  one  near  or  at  the 

34 


530 


PROTOZOA 


opposite  part  of  the  periphery.  This  smaller  body  stains  more  intensely 
than  the  larger  one,  while  the  cytoplasm  of  the  parasite  stains  very 
dimly,  sometimes  showing  only  a  faint  peripheral  rim.  Any  nuclear 
and  cytoplasmic  staining  methods  will  bring  out  these  points  in  Zenker 
fixed  material.  Smears  stain  well  by  Wright  or  the  Nocht-Roman- 
owsky  method.  The  large  cells  containing  the  parasites  are  supposed 
by  Christopher  to  be  the  endothelial  cells  from  the  finest  capillaries. 
Leishman,  Marchand,  Rogers  and  others  think  the  bodies  one  stage 
in  the  life  cycle  of  a  flagellate,  possibly  a  trypanosome,  Rogers  arid 
Marchand  having  seen  flagellates  develop  from  infected  tissue  in  vitro 
in  non-coagulable  blood.  Donovan,  however,  claims  to  have  found 
small  forms  in  the  red  blood  cells  in  the  peripheral  circulation  when 


FIG.  162 


Protozoa  in  a  case  of  tropical  ulcer.  Smear  preparation  from  the  lesion  stained  with  Wright's 
Romanowsky  blood-staining  fluid.  The  ring-like  bodies  with  white  central  portions  and  containing 
a  larger  and  a  smaller  dark  mass  are  the  micro-organisms.  The  dark  masses  in  the  bodies  are 
stained  a  lilac  color,  while  the  peripheral  portions  of  the  bodies  in  typical  instances  are  stained  a 
pale  robin's-egg  blue.  The  very  dark  masses  are  nuclei  of  cells  of  the  lesion.  X  1500  approximately. 
(After  Wright.) 

the  temperature  was  above  103°,  and  his  observation  has  been  con- 
firmed by  Lave  ran  and  Mesnil,  who  believe  the  organism  to  be  a  piro- 
plasma.  Segregation  is  recommended  as  the  best  means  of  eradicating 
the  disease. 

Rabies  (Hydrophobia). 

Rabies  is  an  acute  disease  of  animals,  dependent  upon  a  specific 
virus,  and  communicated  by  inoculation  to  man.  It  is  usually  associated 
with  an  injury,  such  as  the  bite  of  a  dog,  or  the  inoculation  of  the 
broken  surface  with  the  saliva  of  an  animal  affected  with  the  disease. 


,<'.  \HIES 


531 


FIG.  163 


This  is  the  so-called  rabies  of  the  streets.  Wolves,  cats,  foxes,  dogs, 
horses,  cows,  and  deer  may  contract  the  disease;  monkeys,  rabbits,  and 
guinea-pigs  are  all  inoculable  with  it,  as,  indeed,  are  all  warm-blooded 
animals.  Rabies  occurs  in  almost  all  parts  of  the  world;  it  is  most 
common  in  Russia,  France,  and  Belgium;  it  is  not  infrequent  in  Austria 
and  in  those  parts  of  Germany  bordering  on  Russia,  and  in  England.  In 
this  hemisphere  it  is  comparatively  rare,  yet  it  occurs  occasionally  in 
various  parts  of  the  United  States,  in  Mexico  and  South  America.  It 
is  extremely  rare  in  North  Germany,  Switzerland,  Holland,  and  Den- 
mark, owing  to  the  wise  provision  that  all  dogs  shall  be  muzzled;  and 
in  Australia  it  has  not  been  known  to  occur. 

Etiology  and  Pathogenesis. — Until  recently  all  of  the  numerous  re- 
sea  rehes  in  regard  to  the  specific  cause  of  rabies  gave  negative  results. 
The  latest  studies  of  this  disease,  however,  make  it  seem  probable  that 
hydrophobia  may  be  added  to  the  growing  list  of  diseases  caused  by 
protozoa.  In  1903  Negri  described  certain  bodies 
in  the  large  nerve  cells  throughout  the  central 
nervous  system  of  animals  dying  of  this  disease. 
He  found  them  in  largest  numbers  in  animals 
dying  after  the  fourteenth  day  of  the  disease,  and 
especially  numerous  in  the  cells  of  the  gray  matter 
of  Ammon's  horns.  He  did  not  attempt  to  study 
the  bodies  very  minutely.  Besides  demonstrating 
them  in  dogs,  he  found  them  in  rabbits,  in  a  cat, 
and  in  a  human  being.  He  considers  the  bodies 
protozoa  and  the  cause  of  rabies.  In  our  labo- 
ratory we  have  fully  corroborated  Negri 's  results. 
There  is  no  doubt  as  to  the  presence  of  the 
bodies  in  large  numbers  in  animals  in  which  the 
-disease  has  progressed  somewhat  slowly.  In 
animals  dying  quickly  of  fixed  virus  we  have 
also  found  many,  but  the  forms  are  very  small, 
so  that  thev  may  easily  be  overlooked  in  poorly 

i  •*  -\\~     i  11*  ii 

prepared  specimens.  We  have  seen  bodies  small 
enough  to  pass  a  Berkefeld,  thus  accounting  for 
the  positive  results  obtained  by  some  observers  after  the  inoculation  of 
filtrates  obtained  from  Berkefeld  filters.  We  have  found  the  larger  forms 
especially  numerous  in  the  brains  of  guinea-pigs  inoculated  with  street 
virus.  Since  the  discovery  of  these  bodies  Poor,  in  the  Health  Depart- 
ment laboratory,  has  examined  23  cases  of  "street  rabies,"  17  in  dogs, 
3  in  horses,  and  2  in  human  beings.  In  all  of  these  cases  he  demon- 
strated the  presence  of  the  Negri  bodies  in  sections  of  the  central 
nervous  system.  All  of  these  cases  were  subsequently  proven  to  be 
hydrophobic  by  animal  inoculation.  In  a  number  of  suspicious  cases, 
which  were  afterward  proven  to  be  non-rabic,  no  bodies  were  found. 

The  smallest  bodies  in  the  sections  seem  to  be  structureless,  taking 
a  homogeneous  purplish  stain  with  eosin  and  methylene  blue.  As  the 
bodies  become  larger  they  present  a  definite  structure.  A  central  grannie 


Negri  bodies  within  nerve 
cell  of    cornu   ammonis   in 
(Schematic.) 


532  PROTOZOA 

surrounded  by  a  slight  space  appears,  which  may  or  may  not  take  a 
deeper  stain.  In  the  larger  forms  there  are  more  than  one  of  these 
granules.  As  many  as  eight  have  been  seen.  These  may  be  arranged 
more  or  less  regularly  about  a  central  larger  one,  or  scattered  less 
regularly  throughout  the  substance  of  the  body. 

We  have  also  identified  these  bodies  in  smears  of  infected  brains  by 
fixing  the  smear  in  methyl  alcohol,  and  staining  for  twenty-four  hours 
by  the  Nocht-Romanowsky  method  as  recommended  by  Ewing.  The 
bodies  stain  a  robin's-egg  blue  or  lilac,  with  dark-blue,  more  or  less 
regularly  arranged  granules  or  rings,  one  in  the  smaller  bodies  and 
more  in  the  larger,  corresponding  with  the  structure  of  the  bodies  seen 
in  sections.  So  far  no  similar  bodies  have  been  seen  in  control  smears, 
and  it  is  evident  that  this  might  prove  a  reliable  method  for  quick  diag- 
nosis; at  least,  in  cases  where  these  bodies  are  fairly  numerous.  It 
seems  to  us  possible  that  the  bodies  are  protozoa  and  that  they  are 
the  cause  of  rabies.  Their  diagnostic  value  is  certain. 

The  bulk  of  the  toxic  material  outside  of  the  central  nervous  system 
appears  to  be  excreted  in  the  saliva  of  the  submaxillary  gland,  though 
a  certain  small  quantity  may  be  excreted  by  the  other  salivary  glands, 
and  also  by  the  lacrymal  glands,  the  pancreas,  and  the  mammae  of 
rabid  animals.  The  poison  may  also  be  found  in  the  suprarenal  bodies 
and  the  peripheral  nerves.  It  has  not  been  found  in  the  blood,  the 
urine,  or  the  aqueous  humor  of  the  eye;  it  has  been  reported  to  have 
been  found  in  the  foetus. 

That  the  disease  is  due  to  some  form  of  organism  which  has  the 
power  of  multiplying  in  the  tissues  and  of  producing  a  toxic  substance, 
which  appears  to  act  specifically  upon  the  central  nervous  system, 
cannot  be  doubted.  As  in  other  specific  infectious  diseases,  the  virus 
is  transmitted  directly  from  animal  to  animal  through  the  medium  of 
some  fluid  or  secretion;  it  is  now  very  generally  recognized  that  the 
disease  cannot  arise  anew,  as  was  at  one  time  assumed.  In  rabies, 
again,  as  in  other  infectious  diseases,  there  is  a  period  of  incubation 
during  which  the  poison  appears  to  increase  in  quantity. 

The  certainty  with  which  the  disease  may  be  produced  and  its 
severity  have  been  found  to  be  governed  by  three  factors:  (1)  the 
quantity  of  the  rabic  virus  introduced;  (2)  the  point  of  inoculation;  (3) 
the  strength  of  the  virus  as  determined  by  the  kind  of  animal  which 
affords  the  cultivation  ground  for  the  growth  of  the  hypothetical  organ- 
ism. It  is  a  matter  of  common  observation  in  hydrophobia  of  man 
that  slight  wounds  of  the  skin,  of  the  limbs,  and  of  the  back  are  often 
followed  by  the  disease  after  an  extremely  long  period  of  incubation; 
while  in  lacerated  wounds  of  the  tip  of  the  fingers,  where  small  nerves 
are  numerous  or  where  the  muscles  and  nerve  trunks  are  reached,  or 
in  lacerated  wounds  of  the  face,  where  there  is  a  similar  abundance  of 
nerves,  the  period  of  incubation  is  usually  much  shorter  and  the  disease 
generally  much  more  rapid.  Experimental  infection  in  animals  is 
produced  with  the  greatest  certainty  when  the  material  from  the  nerve 
centre  (the  spinal  cord  or  bulb)  of  an  infected  animal  is  injected  into 


RABIES  533 

the  dura  mater  of  the  brain.  It  may  be  produced  almost  as  certainly 
when  the  infection  is  made  into  the  anterior  chamber  of  the  eye  or  into 
the  greater  nerve  trunks.  Intravenous  injection  is  usually  followed  by 
positive  results  in  small  animals,  but  the  larger  animals  do  not  succumb 
to  this  mode  of  inoculation.  Subcutaneous  inoculation  in  animals  is 
uncertain,  because  the  peripheral  nerves  are  not  always  injured;  but 
injection  directly  into  a  mass  of  muscle,  especially  into  parts  which  are 
rich  in  nerves,  almost  invariably  produces  the  disease.  Absorption  of 
the  rabic  poison,  even  from  a  healthy  mucous  surface,  has  been  said 
to  have  taken  place;  and  the  conjunctiva,  the  nasal  and  genital  mucous 
membranes,  and  the  digestive  tract  have  been  noted  as  unabraded  sur- 
faces from  which  this  has  occurred.  The  rapidity  with  which  the  virus 
is  diffused  through  the  body  from  the  point  of  inoculation  in  the  tissues 
seems  to  vary  according  to  the  location  of  the  wound,  but  it  is  always 
comparatively  slow.  It  has  been  found  that  rabbits,  when  etherized 
and  then  presented  to  a  mad  dog  to  be  bitten  on  the  fur,  escape  the 
disease  in  a  very  large  proportion  of  cases,  although  the  teeth  may  have 
passed  well  through  the  skin;  if,  on  the  other  hand,  the  part  presented 
to  the  rabid  dog  be  shaved  before  it  is  bitten,  the  bitten  animals  con- 
tract rabies  in  a  much  larger  proportion  of  cases.  So  in  man,  in  many 
cases  the  rabic  virus  may  be  cleaned  from  the  teeth  by  the  clothing 
which  covers  the  bitten  part  before  they  come  in  contact  with  the  skin. 
From  what  has  been  said  it  is  evident  also  that  when  the  skin  is  thick 
and  the  nerves  few  a  small  quantity  of  virus  may  find  its  way  into  a 
wound,  but  not  penetrate  into  the  nerves,  and  thus  the  person  bitten 
by  a  rabid  animal  may  escape  without  any  ill  effects  beyond  those  due 
to  a  lacerated  wound.  This  will  explain  the  fact  that  only  about  16 
per  cent,  of  the  cases  bitten  by  rabid  animals  appear  to  contract  hydro- 
phobia. 

Preventive  Inoculation  against  Rabies. — The  old  treatment  of  rabies 
consisted  simply  in  encouraging  bleeding  from  the  wound,  or  in  first 
excising  the  wound  and  then  encouraging  bleeding  by  means  of  liga- 
tures, warm  bathing,  cupping-glasses,  etc.;  the  raw  surface  was  then 
freely  cauterized  with  caustic  potash,  nitric  acid,  or  the  actual  cautery. 
It  is  doubtful  whether  the  disease  ever  manifests  itself  after  such  heroic 
treatment  if  the  wound  be  small;  but  when  the  wounds  were  numerous 
or  extensive  the  mortality  from  it  was  still  high.  As  it  was  often  impos- 
sible to  apply  cauterization  to  the  wound  rapidly  or  deeply  enough  to 
ensure  complete  destruction  of  the  virus,  Pasteur  and  others  were 
led  to  study  the  disease  experimentally  in  animals,  with  the  hope  of 
finding  some  means  of  immunization  or  even  cure  through  bacterio- 
logical technique;  these  investigations  finally  resulted  in  the  discovery 
of  methods  of  preventive  inoculation  applicable  to  man. 

Immunization  against  rabies  may  be  effected  in  several  different 
ways.  Pasteur's  treatment  is  based  upon  the  fact  that  rabic  virus  may 
be  attenuated  or  intensified  for  any  animal  at  will.  He  first  observed 
that  the  tissues  and  fluids  taken  from  rabid  animals  varied  considerably 
in  their  virulence.  Then  he  showed  that  the  virus  taken  from  similar 


534  PROTOZOA 

positions — say,  the  cerebrospinial  fluid — had  always  the  same  action 
in  the  same  species;  but  that  fluid  taken  from  an  animal  of  different 
species  was  weaker  or  stronger  as  the  case  might  be.  Thus  the  cerebro- 
spinal  fluid  of  a  series  of  dogs  is  of  constant  strength  and  inoculations 
made  from  dog  to  dog  regularly  produce  death  from  rabies,  the  animals 
passing  through  an  incubation  period  fairly  constant  in  length,  and 
through  a  series  of  similar  symptoms  up  to  death  at  the  expiration  of  the 
same  term.  If,  however,  a  series  of  mortkeys  be  inoculated  the  virus  gradu- 
ally becomes  attenuated,  and  this  attenuation  becomes  more  and  more 
marked  in  successive  inoculations  until  eventually,  after  the  disease  has 
run  a  longer  and  longer  course  in  the  successive  animals,  there  comes  a 
time  at  which  the  virus  is  no  longer  sufficiently  active  to  cause  death. 
If  this  attenuated  fluid  be  now  passed  through  a  series  of  rabbits,  dogs, 
or  guinea-pigs  it  comes  back  to  such  a  strength  that  it  will  kill,  though 
slowly;  then,  however,  its  virulence  gradually  increases  until  the  original 
intensity  is  reached.  If  successive  inoculations  be  made  into  rabbits 
with  fluid,  either  from  the  dog  or  the  monkey,  the  virulence  may  be  so 
exalted  beyond  that  of  the  virus  taken  from  a  street  dog,  in  which  the 
incubation  period  is  from  twelve  to  fourteen  days,  that  at  the  end  of 
the  one  hundredth  passage  the  incubation  period  may  be  reduced  to 
about  six  or  seven  days.  This,  the  strongest  virus  obtainable,  was 
called  by  Pasteur  the  "fixed  virus."  Rabic  virus  appears  also  to 
become  attenuated  under  certain  conditions  of  temperature;  indeed, 
if  it  be  subjected  for  about  an  hour  to  50°  C.,  or  in  half  an  hour 
if  to  60°  C.,  its  activity  is  completely  destroyed.  A  5  per  cent,  solu- 
tion of  carbolic  acid,  acting  for  the  same  period,  exerts  a  similar 
effect,  as  do  likewise  1 : 1000  solutions  of  bichloride  of  mercury,  acetic 
acid,  or  potassium  permanganate.  The  virus  also  rapidly  loses  its 
strength  by  exposure  to  air,  especially  in  sunlight;  when,  however,  pro- 
tected from  heat,  light,  and  air  it  retains  its  virulence  for  a  long  period. 
In  his  earlier  experiments  Pasteur  selected  a  series  of  rabic  poisons 
of  different  strengths,  beginning  with  that  obtained  from  the  spinal 
cord  of  the  monkey — from  the  very  weak  to  the  strongest  that  he  could 
obtain  in  this  animal — then  passing  through  a  similar  series  obtained 
during  the  process  of  exaltation  of  the  virus  by  passage  through  the 
rabbit.  By  inoculating  dogs  subcutaneously  with  virus  taken  from  a 
series  commencing  with  the  weakest  taken  from  a  monkey,  and  grad- 
ually working  up  to  that  obtained  from  the  rabbit — from  the  earliest 
to  the  latest  in  the  series — the  animals  become  immune  not  only  against 
subcutaneous  injection,  but  against  subdural  infection  with  fixed  virus, 
and  also  against  the  bite  of  rabid  dogs.  Such  a  method  as  this,  how- 
ever, had  several  disadvantages,  and  was  not  absolutely  certain  in  its 
action,  as  only  fifteen  out  of  twenty  dogs  were  completely  protected. 
Pasteur,  therefore,  assisted  by  Chamberland  and  Roux,  devised  a  more 
trustworthy  and  accurate  method,  in  which  he  utilized  the  fact  that  the 
cord  of  a  rabic  animal  when  kept  under  certain  conditions  loses  its  viru- 
lence in  fourteen  days.  A  series  of  cords  cut  into  short  segments,  which 
were  held  in  series  by  the  dura  mater,  were  suspended  in  sterile  glass 


RABIES  535 

flasks  plugged  with  cotton  stoppers,  and  containing  a  quantity  of  some 
hygroscopic  material,  such  as  caustic  potash;  and  the  whole  was  kept 
at  a  temperature  of  about  22°  (\  The  cord  when  taken  out  at  the  end 
of  the  first  twenty-four  hours  was  found  to  be  almost  as  active  as  the 
fresh  untreated  cord;  that  removed  at  the  end  of  forty-eight  hours  wa^ 
slightly  less  active  than  that  removed  twenty-four  hours  previously; 
and  the  diminution  in  virulence,  though  gradual,  progressed  regularly 
and  surely  until,  as  already  noted,  at  the  end  of  the  fourteenth  or  fif- 
teenth day  the  virus  was  inactive.  An  emulsion  of  the  cord  of  the  last 
day  was  made,  and  a  certain  quantify  injected  into  a  dog  that  had  been 
bitten;  this  was  followed  by  an  injection  of  an  emulsion  of  a  thirteenth- 
day  cord,  and  so  on  until  the  animal  had  been  injected  with  a  perfectly 
fresh  and,  therefore,  extremely  active  cord,  corresponding  to  the  fixed 
virus.  Animals  treated  in  this  way  were  now  found  to  be  absolutely 
protected,  even  against  subdural  inoculation  with  considerable  quantities 
of  the  most  virulent  virus;  and  thus  Pasteur's  protective  inoculation 
against  rabies  became  an  accomplished  fact.  As  it  would  be  impossible, 
however,  or  very  undesirable,  to  inject  any  but  persons  who  had  actually 
been  bitten  by  a  rabid,  or  presumably  rabid,  animal,  Pasteur  continued 
his  experiments,  in  order  to  see  whether  it  would  not  be  possible  to 
cure  a  patient  already  bitten.  He  carried  on,  therefore,  a  series  of 
experiments  which  led  to  the  discovery  that  if  the  process  of  inoculation 
be  begun  within  five  days  of  the  bite  in  animals  in  which  the  incubation 
period  was  at  least  fourteen  days,  almost  every  animal  bitten  can  be 
saved ;  and  that  even  if  the  treatment  be  commenced  at  a  longer  interval 
after  the  bite  a  certain  proportion  of  recoveries  can  be  obtained.  Thus 
the  application  of  this  method  of  treatment  to  the  human  subject  was 
not  tried  until  it  had  been  proved  in  animals  that  such  protection  could 
be  obtained,  and  that  such  protection  would  last  for  at  least  two  years, 
and  probably  longer. 

The  chance  of  success  in  the  human  subject  appears  to  be  even 
greater  than  in  the  dog  or  rabbit,  seeing  that  on  account  of  the  resist- 
ance offered  by  the  human  tissues  to  the  virus  the  period  of  incubation 
is  comparatively  prolonged;  very  rarely,  if  ever,  does  an  outbreak  of  the 
disease  in  man  occur  before  an  interval  of  at  least  fifteen  days.  The 
first  symptoms  usually  appear  in  the  fifth  or  sixth  week,  sometimes  not 
until  the  third  month;  exceptionally  the  incubation  period  has  lasted 
for  a  year.  Thus  there  is  an  opportunity  of  obtaining  immunity  by 
beginning  the  process  of  vaccination  soon  after  the  bite  has  been  inflicted, 
the  protection  being  complete  before  the  incubation  period  has  passed. 
In  his  earlier  experiments  Pasteur  injected  on  each  succeeding  day 
emulsions  from  a  cord  dried  for  one  day  less  until  cords  dried  five  days 
were  reached;  but  later  he  used  those  dried  for  only  three  days.  This 
was  the  "simple"  ten-day  method.  It  was  soon  evident  that  although 
this  method  was  efficacious  where  the  wounds  were  not  severe,  and 
were  confined  to  parts  in  which  the  nerve  supply  was  not  extensively 
interfered  with,  it  was  often  quite  inadequate  in  serious  cases,  as  of 
wounds  about  the  face,  or  of  wounds  inflicted  by  a  mad  wolf,  the  virus 


636  PROTOZOA 

of  which  is  more  active  and  the  lesions  made  more  severe  than  that  of 
the  rabid  dog  of  the  streets.  In  these  latter  cases  the  injections  which, 
in  the  simple  treatment,  are  spread  over  five  days  are  made  in  three 
days;  then,  on  the  fourteenth  day,  a  fresh  series  of  injections,  or,  rather 
repetitions,  is  begun,  which  lasts  until  the  twenty-first  day.  This  is 
the  "  intensive  method."  In  the  technique  of  the  treatment,  which  is  the 
same  in  both  methods,  a  small  portion  (about  1  cm.)  of  the  desiccated 
cord  is  rubbed  up  thoroughly  with  about  four  or  five  times  its  bulk  of 
bouillon  until  a  complete  emulsion  is  made;  this  is  then  injected  by 
means  of  a  syringe  holding  several  cubic  centimetres,  first  on  one  side 
of  the  hypochondriac  region  and  then,  the  following  day,  on  the  other, 
and  so  on  alternately,  to  avoid  irritation.  With  the  observance  of 
thorough  asepsis  no  local  reaction  to  speak  of  takes  place,  nor  are 
abscesses  ever  formed.  The  results  of  Pasteur's  .method  of  protective 
inoculation,  as  recorded  in  the  reports  issued  in  the  Annales  de  I'lnstitut 
Pasteur  and  those  of  other  antirabic  institutes  in  Italy,  Russia,  Rou- 
mania,  etc.,  are  very  favorable.  Since  1886,  when  the  treatment  was 
first  commenced  at  the  Pasteur  Institute  in  Paris,  upward  of  20,000 
persons  bitten  by  rabid,  or  presumably  rabid,  animals  have  received 
preventive  inoculations,  with  a  mortality  of  only  0.5  of  1  per  cent. 
The  mortality  of  those  bitten  on  the  face  or  head  was  1.25  per  cent,  of 
those  bitten  on  the  hand;  it  was  0.75  of  1  percent,  of  those  bitten  on 
other  parts  of  the  body,  a  little  over  0.25  of  1  per  cent.  As  a  rule,  only 
those  persons  are  treated  who  have  been  bitten  on  the  face  or  hand 
or  whose  clothes  have  been  lacerated  so  that  the  virus  has  passed  into 
the  wounds.  Ordinarily,  a  certificate  from  a  physician  or  a  veterinarian 
that  the  animal  was  rabid  is  required  before  vaccination;  but  if  the 
animal  cannot  be  found  or  the  wounds  are  severe,  vaccination  is  per- 
formed without  it.  Taking  only  the  cases  in  which  rabies  has  been 
confirmed  in  the  animal  by  a  veterinary  surgeon,  the  mortality  of  the 
cases  treated  at  the  Pasteur  Institute  in  Paris  is  only  0.6  per  cent. — a 
proof,  it  would  seem,  of  the  trustworthiness  of  the  statistics.  In  view 
of  this  fact  there  can  no  longer  be  any  doubt  of  the  value  of  Pasteur's 
antirabic  treatment.  It  has  been  stated  by  some  that  the  percentage 
of  persons  killed  by  the  bites  of  rabid  animals  is  inconsiderable;  but 
according  to  the  reliable  statistics  of  Leblanc,  from  1878  to  1883,  out 
of  515  persons  bitten  in  Paris,  83  died  of  hydrophobia,  a  mortality  of 
16  per  cent.;  most  authorities  place  the  mortality  at  a  much  higher 
figure.  Extensive  bites  on  the  face  and  head  are  considered  to  be  par- 
ticularly dangerous;  the  mortality  of  these  is  said  to  be  at  least  80 
per  cent.  The  bites  of  wolves  seem  to  be  more  fatal  than  the  bites 
of  dogs  or  other  animals;  the  mortality  of  these,  in  spite  of  the  most 
intensive  treatment,  is  stated  to  be  still  10  per  cent.,  as  against  a 
previous  mortality,  without  specific  treatment,  of  40  to  60  per  cent. 
But  even  Pasteur's  antirabic  treatment  appears  to  be  unavailable 
when  symptoms  of  the  disease  have  manifested  themselves.  Our 
results  in  the  New  York  Department  of  Health  have  been  very  en- 
couraging. 


RABIES  537 

Other  methods  of  immunization  against  rabies  have  been  proposed 
by  different  investigators.  But  all  of  these  methods  have  proved  on 
trial  to  be  unsatisfactory  and  unreliable,  beside  being  not  devoid  of 
danger.  As  early  as  1889,  Babes  and  Lepp  conceived  the  idea  that  it 
might  be  possible  by  means  of  the  blood  to  transmit  conferred  immunity 
against  rabies  from  one  animal  to  another;  but  although  the  success 
of  these  investigators  was  not  great,  Tizzoni  and  Schwartz,  and  later 
Tizzoni  and  Centanni,  worked  out  a  method  of  serum  inoculation  and 
protection  in  rabies  which  is  worthy  of  attention.  In  this  method  not 
the  rabic  poison  itself  but  the  protective  material  formed  is  injected 
into  the  tissues.  These  observers  showed  that  the  serum  of  inoculated 
animals  is  capable  of  destroying  the  pathogenic  power  of  the  rabic 
virus — not  only  when  mixed  with  it  before  injection,  but  when  injected 
simultaneously  or  within  twenty-four  hours  after  the  introduction  of 
the  virus  into  the  body.  This  serum  treatment  of  rabies  is  still  in  the 
experimental  stage.  We  ourselves  have  had  no  experience  with  it, 
nor  has  it  been  adopted  in  Paris,  or,  so  far  as  we  know,  in  other  places. 
It  is  quite  possible  that  others  will  not  obtain  such  good  results  as  the 
authors  of  the  treatment,  or  that  it  may  not  prove  so  efficacious  in 
the  treatment  of  man  as  it  has  been  found  to  be  in  experimental 
work. 

The  Cauterization  of  Infected  Wounds. — It  is  commonly  believed  that 
unless  a  cautery  is  used  within  an  hour  after  infection  by  a  suspected 
animal  it  is  useless  to  apply  it.  This  belief  is  held  by  physicians  in 
general,  and  also,  apparently,  so  far  as  the  literature  seen  by  me  indi- 
cates, by  those  familiar  with  rabies.  For  this  reason  physicians  when 
applying  a  cautery  later  than  an  hour  after  infection  do  so  largely  as 
a  matter  of  form,  for  its  moral  effect  on  the  patient,  and  so  the  appli- 
cation is  not  thorough,  and  in  consequence  not  effectual.  There  is  no 
evidence  to  show  that  the  cautery  is  useless  after  an  hour;  no  systematic 
investigations  have  been  published,  so  far  as  we  know,  to  prove  the 
point  one  way  or  the  other. 

We  know  that  the  virus  of  rabies  is  not  carried  into  the  system  by 
the  blood,  but  through  the  nervous  system.  Dr.  Follen  Cabot  carried 
out  an  extensive  series  of  experiments  in  our  laboratory  upon  guinea- 
pigs  which  showed :  (1)  That  91  per  cent,  of  guinea-pigs  can  be  prevented 
from  developing  rabies  if  the  wounds  be  cauterized  with  chemically 
pure  nitric  acid  at  the  end  of  twenty-four  hours  from  the  time  of  infec- 
tion, probably  a  larger  percentage  if  the  cautery  be  used  earlier.  (2) 
That  fuming  nitric  acid  is  more  effectual  than  the  actual  cautery  of 
pure  nitrate  of  silver.  (3)  That  some  degree  of  benefit  is  derived  from 
thoroughly  opening  and  swabbing  out  an  infected  wound  within  twenty- 
four  hours  from  the  time  of  infection  when  no  cautery  is  used.  I  believe 
that  he  demonstrated  that  in  cases  in  which  the  Pasteur  treatment 
cannot  be  applied  great  benefit  may  be  derived  from  the  correct  use 
of  cauterization,  even  twenty-four  hours  after  infection,  and  that  even 
in  cases  in  which  the  Pasteur  treatment  can  be  given,  an  early  cauteriza- 
tion will  be  of  great  assistance  as  a  routine  practice,  and  should  be 


538  PROTOZOA 

very  valuable,  as  the  Pasteur  treatment  is  frequently  delayed  several 
days,  for  obvious  reasons,  and  does  not  always  protect.  In  the  case  of 
small  wounds  all  the  treatment  probably  indicated  will  be  thorough 
cauterization  with  nitric  acid  within  twelve  hours  from  the  time  of 
infection.  Our  experience  in  dealing  with  those  bitten  by  rabid  animals 
goes  to  show  that  physicians  do  not  appreciate  the  value  of  thorough 
cauterization  of  the  infected  wounds. 

Pasteur  Treatment  by  Mail. — For  several  years  we  have  made  a  prac- 
tice of  sending  the  treatment  by  mail  when  the  patients  could  not  go 
for  treatment.  The  results  have  been  good. 

But  far  more  important  than  any  treatment,  curative  or  preventive, 
for  hydrophobia  in  man  is  the  prevention  of  rabies  in  dogs,  through 
which  this  disease  is  usually  conveyed.  Were  all  dogs  under  legislative 
control  and  the  compulsory  wearing  of  muzzles  rigidly  enforced  where 
rabies  prevails,  hydrophobia  would  soon  become  an  almost  unknown 
disease.  This  fact  has  been  amply  demonstrated  by  the  statistics  of 
rabies  in  countries  where  such  laws  are  now  in  force. 


APPENDIX. 


Aggressins. 

A  FURTHER  contribution  has  recently  been  made  to  the  problems  of 
virulence  and  immunity  in  the  form  of  the  "  aggressin  theory  "  of  Bail.1 
Apparently  it  grew  out  of  an  attempt  to  explain  the  so-called  "  phe- 
nomenon of  Koch  " — an  observation  made  years  ago  by  Koch — to  the 
effect  that  tuberculous  animals  when  inoculated  intraperitoneally  with 
a  fresh  culture  of  tubercle  bacilli  succumb  quickly  to  an  acute  attack 
of  the  disease,  the  resulting  exudate  containing  almost  exclusively 
lymphocytes.  Bail  found  that  if  tubercle  bacilli,  together  with  steril- 
ized tuberculous  exudate,  were  injected  into  healthy  guinea-pigs, 
the  animal  died  very  suddenly — i.  e.,  in  twenty-four  hours  or  there- 
abouts. The  exudate  alone  had  no  appreciable  effect  on  the  animal, 
while  inoculation  with  tubercle  bacilli  alone  produced  death  in  a 
number  of  weeks.  He  therefore  concludes  that  there  is  something  in 
the  exudate  that  allows  the  bacilli  to  become  more  aggressive,  and 
hence  has  called  this  hypothetical  substance  "  aggressin/'  He  thinks 
it  is  an  endotoxin  liberated  from  the  bacteria  as  a  result  of  bacteriolysis 
and  that  it  acts  by  paralyzing  the  polynuclear  leukocyte,  thereby  pre- 
venting phagocytosis.  Heating  the  exudate  to  60°  C.  increases  its 
aggressive  properties  rather  than  diminishes  them  and  small  doses  act 
relatively  more  strongly  than  larger  ones.  These  facts  he  explains  by 
assuming  the  presence  of  two  properties,  one  that  prevents  rapid  death 
is  thermolabile  and  acts  feebly  in  small  doses,  and  one  that  favors 
rapid  death  and  is  thermostabile.  He  assumes  that  in  a  tuberculous 
animal  the  tissues  are  saturated  with  the  aggressin  and  when  fluid  col- 
lects in  the  body  cavities,  as  it  does  on  injection  of  tubercle  bacilli,  it 
contains  large  quantities  of  aggressin,  which  prevents  migration  of  the 
polynuclear  le'.ikncytivs  but  not  of  the  lymphocytes,  and  hence  allows  the 
bacilli  to  develop  freely,  producing  acute  symptoms.  In  the  peritoneal 
cavity  of  the  normal  animal  injected  with  tubercle  bacilli,  on  the  other 
hand,  are  large  numbers  of  polynuclear  leukocytes  which  engulf  the 
bacilli,  thus  inhibiting  their  rapid  development,  there  being  here  no 
aggressin  to  prevent  phagocytosis. 

This  theory  has  been  applied  to  a  number  of  infections,  includ- 
ing typhoid,  cholera,  dysentery,  chicken  cholera,  pneumonia,  and 
staphylococcus  infections.  In  all  similar  results  have  been  obtained  as 

1  Wiener  klin.  Woch.,  1905,  No.  9.  Ibid.,  1905,  Nos.  14,  16,  17.  Berliner  kliri.  Woch.,  1905, 
N...  15.  Zeit.  f.  Hyg.,  1905,  vol.  i.  No.  3.  Arch.  f.  Hyg.,  vol.  Hi.  pp.  272  and  411. 


540  APPENDIX 

with  tubercle  bacilli.  When  exudates,  produced  by  virulent  cultures  of 
these  various  organisms  and  properly  sterilized,  are  injected  with  fresh 
cultures  into  an  animal  death  occurs  in  much  shorter  time  than  when 
the  organisms  alone  are  injected. 

Moreover,  it  has  been  possible  to  immunize  animals  against  these 
various  infections  by  repeated  injections  of  the  aggressin  in  the  form  of 
exudates.  This  results  in  the  formation  of  an  "  antiaggressin,"  which 
opposes  the  action  of  the  aggressin,  thereby  enabling  the  leukocytes  to 
take  up  the  bacteria  and  thus  to  protect  the  animal.  This  has  been 
done  in  staphylococcus,  dysentery,  typhoid,  cholera,  pneumococcus, 
and  chicken  cholera  infections  in  animals.  In  addition  a  very  marked 
agglutinative  property  of  the  blood  is  acquired  for  the  bacteria  in  the 
animals  so  immunized. 


Experiments   Devised  by  Ehrlich  to   Show  the  Nature   of 

Hsemolysins. 

In  order  to  give  the  student  an  idea  of  the  methods  employed  by 
Ehrlich  in  developing  his  doctrine  of  immunity,  the  classical  series  of 
experiments  made  to  show  the  nature  of  hsemolysins  are  here  reproduced. 

Ehrlich  asked  himself  two  questions :  (1)  What  relation  does  the  hsemo- 
lytic  serum  or  its  two  active  components,  immune  body  and  complement, 
bear  to  the  cell  to  be  dissolved  ?  (2)  On  what  does  the  specificity  of  this 
hsemolytic  process  depend?  He  made  his  experiments  with  a  hsemo- 
lytic  serum  that  had  been  derived  from  a  goat  treated  with  the  red  cells 
of  a  sheep.  This  serum,  therefore,  was  hsemolytic  specifically  for  sheep 
blood  cells — i.  e.,  it  possessed  increased  solvent  properties  exclusively  for 
sheep  blood  cells.  Basing  his  reasoning  on  the  side-chain  theory,  Ehrlich 
argued  as  follows:  "If  the  hsemolysin  is  able  to  exert  a  specific  solvent 
action  on  sheep  blood  cells,  then  either  of  its  two  factors,  the  immune 
body  or  the  alexin  (complement)  of  normal  serum,  must  possess  a 
specific  affinity  for  these  red  cells."  To  show  this  he  devised  the  follow- 
ing series  of  experiments: 

EXPERIMENT  1. — Ehrlich  and  Morgenroth,  as  already  said,  experi- 
mented with  a  serum  that  was  specifically  hsemolytic  for  sheep  blood 
cells.  They  made  this  inactive  by  heating  to  55°  C.,  so  that  then  it 
contained  only  the  substance  sensibilatrice  (immune  body).  Next  they 
added  a  sufficient  quantity  of  sheep  red  blood  cells,  and  after  a  time 
centrifuged  the  mixture.  They  were  now  able  to  show  that  the  red 
cells  had  combined  with  all  the  substance  sensibilatrice,  and  that  the 
supernatant  clear  liquid  was  free  from  the  same.  In  order  to  prove 
that  such  was  the  case  they  proceeded  thus :  To  some  of  the  clear  cen- 
trifuged fluid  they  added  more  sheep  red  cells;  and,  in  order  to  reacti- 
vate the  serum,  a  sufficient  amount  of  alexin  in  the  form  of  normal 
serum  was  also  added.  The  red  cells,  however,  did  not  dissolve — there 
was  no  substance  sensibilatrice.  The  next  point  to  prove  was  that 
this  substance  had  actually  combined  with  the  red  cells.  The  red 


EXPERIMENTS  TO  SHOW  THE  NATURE  OF  HAMOLYSINS    541 

cells  which  had  been  separated  by  the  centrifuge  were  mixed  with  a 
little  normal  salt  solution  after  freeing  them  as  much  as  possible  from 
fluid.  Then  a  little  alexin  in  the  form  of  normal  serum  was  added. 
After  remaining  thus  for  two  hours  at  37°  C.  these  cells  had  all 
dissolved. 

In  this  experiment,  therefore,  the  red  cells  had  combined  with  all 
the  substance  sensibilatrice,  entirely  freeing  the  serum  of  the  same. 
That  the  action  was  a  chemical  one,  and  not  a  mere  absorption,  was 
shown  by  the  fact  that  red  blood  cells  of  other  animals,  rabbits  or  goats 
for  example,  exerted  no  combining  power  at  all  when  used  instead  of 
the  sheep  cells  in  the  above  experiment.  The  union  of  these  cells,  more- 
over, is  such  a  firm  one  that  repeated  washing  of  the  cells  with  normal 
salt  solution  does  not  break  it  up. 

The  second  important  question  solved  by  these  authors  was  this: 
What  relation  does  the  alexin  bear  to  the  red  cells?  They  studied  this 
by  means  of  a  series  of  experiments  similar  to  the  preceding. 

EXPERIMENT  2. — Sheep  blood  was  mixed  with  normal — i.  e.,  not 
hsemolytic  goat  serum.  After  a  time  the  mixture  was  centrifuged  and 
the  two  portions  tested  with  the  substance  sensibilatrice  to  determine  the 
presence  of  alexin.  It  was  found  that  in  this  case  the  red  cells  acted 
quite  differently.  In  direct  contrast  to  their  behavior  toward  the  sub- 
stance sensibilatrice  in  the  first  experiment,  they  now  did  not  combine 
with  even  the  smallest  portion  of  alexin,  and  remained  absolutely 
unchanged. 

EXPERIMENT  3. — The  third  series  of  experiments  was  undertaken 
to  show  what  relations  existed  between  the  blood  cells  on  the  one  hand 
and  the  substance  sensibilatrice  and  the  alexin  on  the  other,  when  both 
were  present  at  the  same  time,  and  not,  as  in  the  other  experiments,  when 
they  were  present  separately.  This  investigation  was  complicated  by  the 
fact  that  the  specific  immune  serum  very  rapidly  dissolves  the  red  cells 
for  which  it  is  specific,  and  that  any  prolonged  contact  between  the 
cells  and  the  serum,  in  order  to  effect  binding  of  the  substance  sensi- 
bilatrice, is  out  of  the  question.  Ehrlich  and  Morgenroth  found  that 
at  0°  C.  no  solution  of  the  red  cells  by  the  hremolytic  serum  takes  place. 
They  therefore  mixed  some  of  their  specific  hsemolytic  serum  with  sheep 
blood  cells,  and  kept  this  mixture  at  0°  to  3°  C.  for  several  hours.  No 
solution  took  place.  They  now  centrifuged  and  tested  both  the  sedi- 
mented  red  cells  and  the  clear  supernatant  serum.  It  was  found  that 
at  the  temperature  0°  to  3°  C.  the  red  cells  had  combined  with  all  of  the 
substance  sensibilatrice,  but  had  left  the  alexin  practically  untouched. 

It  still  remained  to  show  the  relation  of  these  two  substances  to  the 
red  cells  at  higher  temperatures.  At  37°  to  40°  C.,  as  already  mentioned, 
haemolysis  ocpurs  rapidly,  beginning  usually  within  fifteen  minutes. 
It  was  possible,  therefore,  to  leave  the  cells  and  serum  in  contact  for 
not  over  ten  minutes.  Then  the  mixture  was  centrifuged  as  before. 
The  sedimented  blood  cells  mixed  with  normal  salt  solution  showed 
haemolysis  of  a  moderate  degree.  The  solution  became  complete  when 
a  little  normal  serum  \va>  added.  The  supernatant  clear  fluid  separated 


542  APPENDIX 

by  the  centrifuge  did  not  dissolve  sheep  red  cells.  On  the  addition, 
however,  of  the  substance  sensibilatrice  it  dissolved  them  completely. 

The  addition  of  red  cells  in  the  experiments  was  always  in  the  form 
of  a  5  per  cent,  mixture  or  suspension  in  0.85  per  cent. — i.  e.,  isotonic, 
salt  solution. 

The  significance  of  the  last  of  the  above-cited  experiments  is,  accord- 
ing to  Ehrlich,  at  once  apparent.  It  is  that  the  substance  sensibilatrice 
possesses  one  combining  group  with  an  intense  affinity  (active  even  at 
0°  C.)  for  the  red  cell,  and  a  second  group  possessing  a  weaker  affinity 
(one  requiring  a  higher  temperature)  for  the  alexin. 

So-called  Ultramicroscopic  Examinations. 

The  apparatus  constructed  by  Siedentopf  and  Zsigmondy1  makes 
visible  in  colloidal  solutions  very  minute  particles,  which  heretofore 
could  not  be  seen  even  with  the  highest  magnifications.  Particles  as 
small  as  a  few  microns  are  thus  rendered  visible. 

FIG.  164 


Virulent  diphtheria  bacilli.     Cultures  two  days  old.     Unstained.     X  2400.      (After  Siebert.) 

This  increased  power  in  microscopic  analysis  is  made  possible  by  focal 
lateral  illumination  of  the  object  to  be  examined.  The  greater  the 
difference  between  the  refractive  index  of  the  particles  colloidally  dis- 
solved and  the  fluid  which  surrounds  them,  the  brighter  will  be  the 
appearance  of  the  particles,  and,  therefore,  the  more  readily  visible. 
The  illumination  for  the  purpose  is  most  intense,  and  furnished  by  an 
electric  arc  lamp. 

The  microscopic  field,  as  will  be  seen  by  the  photogram  herewith, 
is  dark;  the  objects  which  refract  the  light  show  as  brightly  illuminated, 

1  Annalen  der  Physik,  4te  Folge,  Band  10. 


ST&;<)MYI.\  /    l>r/  17.1  AM)  ITS  RELATION  TO  YELLOW  FEVER     543 

sharply  defined  pictures,  in  which  the  black  margin  corresponds  to  the 
contour  of  the  object.  The  illuminated  portion  is  surrounded  by  a  fine 
dark  /.one,  this  in  turn  by  alternate  bright  and  dark  zones,  in  which  the 
illumination  rapidly  decreases. 

With  a  suitable  apparatus  both  stained  and  unstained  bacteriological 
specimens  can  be  examined. 

Variation  in  Susceptibility  of  Guinea-pigs  to  Diphtheria  Toxin. 

Smith  has  recently  shown  that  a  small  percentage  of  guinea-pigs  shows 
a  marked  resistance  to  the  poisonous  effect  of  diphtheria  toxin.  This 
refractory  condition  is  inherited  from  the  mother.  This  fact  has  to  be 
considered  in  the  testing  of  toxin  and  antitoxin. 

Diplobacillus  of  Morax-Axenfeld, 

This  organism  appears  to  be  the  exciting  agent  of  a  fairly  frequent 
and  peculiar  infectious  disease  affecting  only,  so  far  as  known,  the 
human  conjunctiva.  The  organism  was  discovered  independently  by 
Morax  and  Axenfeld  in  1896.  It  is  not  pathogenic  for  animals.  The 
usual  clinical  picture  is  that  of  a  "blepharoconjunctivitis."  The  sub- 
jective symptoms  are  relatively  slight. 

Examination  of  the  conjunctival  secretion  shows  the  characteristic 
bacilli.  These  are  arranged  mostly  in  twos,  though  long  and  short  chains 
are  also  found.  The  bacilli  average  2/u  long  and  \fJ.  wide,  though  smaller 
diplobacilli  are  often  seen,  probably  younger  forms.  The  ends  of  the 
organisms  are  slightly  rounded,  and  usually  of  the  same  thickness  as  the 
rest  of  the  cell.  The  line  of  demarcation  between  the  individuals  is 
distinct.  The  bacilli  are  not  stained  by  Grain's  method  and  have  no 
distinct  capsules.  The  organism  grows  only  at  near  the  body  temperature 
and  best  on  solidified  blood  serum  or  serum  agar.  The  medium  must 
be  alkaline. 

Bacteria  in  Ice. 

Water  when  it  turns  to  ice  destroys  a  considerable  percentage  of  the 
vegetative  forms  of  bacteria.  Spores  are  not,  as  a  rule,  injured.  Ice 
six  months  or  more  after  freezing  contains  very  few  bacteria  even  when 
made  from  polluted  water.  Not  more  than  10  per  cent,  of  typhoid 
bacilli  survive  freezing.  At  the  end  of  one  week  not  more  than  1  per 
cent,  remain  alive.  Not  one  in  one  thousand  survive  for  one  month,  and 
none  for  more  than  six  months.  The  colon  group  of  bacilli  are  a  little 
more  hardy  than  the  typhoid  bacilli. 

Stegomyia  Fasciata  and  its  Relation  to  Yellow  Fever.1 

The  experiments  made  by  Reed,  Carroll,  and  Agramonte  make  it 
certain  that  yellow  fever  was  transmitted  by  a  mosquito  (Stegomyia 

1  This  account  IB  taken  from  the  article  by  Agramonte  in  Laboratory  Work  with  Mosquitoes,  by 
Berkeley. 


544  APPENDIX 

fasciata),  in  the  same  way  that  malaria  is  transmitted  by  Anopheles.  (See 
p.  442.)  The  name  Stegomyia  was  suggested  by  the  English  entomologist 
Theobald,  who  separated  this  genus  from  the  genus  Culex,  with  which 
it  was  formerly  classed.  The  salient  characteristics  of  Stegomyia  are : 
(1)  the  palpi  in  the  male  are  as  long,  or  nearly  as  long,  as  the  proboscis;  in 
the  female  the  palpi  are  uniformly  less  than  one-half  as  long;  (2)  the  legs 
are  destitute  of  erect  scales ;  (3)  the  thorax  is  marked  with  lines  of  silvery 
scales.  Stegomyia,  or  at  least  Stegomyia  fasciata,  is  spread  over  a  wide  range 
of  territory,  embracing  many  varieties  of  climate  and  natural  conditions. 
It  has  been  found  as  far  north  as  Charleston,  S.  C.,  and  as  far  south  as 
Rio  de  la  Plata.  There  is  no  reason  to  believe  that  it  may  not  be  present 
at  some  time  or  other  in  any  of  the  intermediate  countries.  In  the  United 
States  specimens  of  Stegomyia  fasciata  have  been  captured  in  Georgia, 
Louisiana,  South  Carolina,  and  Eastern  Texas.  The  island  of  Cuba 
is  overrun  with  this  insect.  The  fact  that  Stegomyia  fasciata  has  been 

FIG.  165 


Adult  female  Stegomyia  fasciata.    (Drawn  by  Agramonte.) 

known  to  exist  at  various  times  in  Spain  and  other  European  countries 
may  account  for  the  spread  of  yellow  fever  which  has  occurred  there 
once  or  twice  in  former  times;  the  same  may  be  said  of  the  countries 
farther  north  in  the  United  States,  where  Stegomyia  fasciata  has  not 
yet  been  reported,  but  which  have  suffered  from  invasions  of  yellow 
fever. 

Brackish  water  is  unsuited  for  the  development  of  Stegomyia  larvae. 
The  species  of  Stegomyia  fasciata  seems  to  select  any  deposit  of  water 
which  is  comparatively  clean.  The  defective  drains  along  the  eaves  of 
tile  roofs  are  a  favorite  breeding  place  in  Havana  and  its  suburbs;  in- 
doors they  find  an  excellent  medium  in  the  water  of  cups  of  tin  or  china 
into  which  the  legs  of  tables  are  usually  thrust  to  protect  the  contents 
from  the  invasion  of  ants,  a  veritable  pest  in  tropical  countries.  The 
same  may  be  said  of  shallow  traps,  where  the  water  is  not  frequently 
disturbed. 

Like  other  Cidicidce,  it  prefers  to  lay  at  night.  It  is  eminently  a  town 
insect,  seldom  breeding  far  outside  of  the  city  limits.  Agramonte  never 


STEGOMYIA  FASCIATA  AND  ITS  RELATION  TO  YELLOW  FEVER     545 

found  Stegomyia  fasciata  resting  under  bushes,  in  open  fields,  or  in  the 
woods;  this  fact  explains  the  well-founded  opinion  that  yellow  fever  is 
a  domiciliary  infection. 

The  question  of  hibernation  in  the  larval  stage  is  important.  Agra- 
monte  failed  to  get  larvae  that  could  resist  freezing  temperature,  and 
found  that  in  the  case  of  Stegomyia  fasciata  this  degree  of  cold  was 
invariably  fatal. 

The  possibility  of  their  being  capable  of  life  outside  their  natural 
element  must  also  be  considered  from  an  epidemiological  point  of  view. 
The  dry  season  in  the  countries  where  this  species  seems  to  abound  is 
never  so  prolonged  as  completely  to  dry  up  the  usual  breeding  places. 
Experimentally,  adult  larvae  removed  from  the  water  and  placed  over- 
night upon  moist  filter  paper  could  not  be  revived  the  following  morning. 

The  question  of  the  life  period  of  the  female  insect  is  of  the  greatest 
importance  when  we  come  to  consider  the  apparently  long  interval 
which  at  times  has  occurred  between  the  stamping  out  of  an  epidemic 
of  yellow  fever  and  its  new  outbreak  without  introduction  of  new  cases. 
The  fact  is  that  Stegomyia  fasciata  is  a  long-lived  insect;  one  individual 
was  kept  by  Agramonte  in  a  jar  through  March  and  April  into  May  for 
seventy-six  days  after  hatching  in  the  laboratory. 

It  was  definitely  shown  by  the  experiment  of  the  Army  Medical  Board 
upon  non-immune  persons  that  a  period  of  at  least  twelve  days  at  a 
temperature  of  about  83°  F.  was  necessary  before  the  infected  insect 
could  transmit  the  germ  of  yellow  fever  from  the  sick;  later  on  a  mos- 
quito which  reached  the  age  of  seventy  days,  in  the  hands  of  Dr.  Carroll, 
was  able  to  produce  a  case  of  yellow  fever  by  stinging  an  American 
soldier  fifty-seven  days  after  it  became  infected. 

These  mosquitoes  bite  principally  in  the  late  afternoon,  though  they 
may  be  incited  to  take  blood  at  any  hour  of  the  day.  They  are  abundant 
from  March  to  September,  and  even  in  November  Agramonte  was  able 
to  capture  them  at  will  in  his  office  and  laboratory. 

The  mosquito  is  generally  believed  to  be  incapable  of  long  flights 
unless  very  materially  assisted  by  the  wind.  At  any  rate,  the  close  study 
of  the  spread  of  infection  of  yellow  fever  shows  that  the  tendency  is  for 
it  to  remain  restricted  within  very  limited  areas,  and  that  whenever  it 
has  travelled  far  beyond  this  the  means  afforded  (railway  cars,  vessels, 
etc.)  have  been  other  than  the  natural  flight  of  the  insect. 

Experimentally,  it  has  been  found  that  the  infected  mosquito  must 
be  kept  at  about  80°  to  85°  F.  for  twelve  days  after  having  bitten  a  yellow 
fever  patient.  This  is  necessary  in  order  to  enable  the  parasites  to  per- 
form the  evolutions  in  the  body  of  the  mosquito  that  will  render  them 
capable  of  reproducing  the  disease.  In  winter  insects  kept  at  this 
temperature  have  failed  to  infect  even  after  eighteen  days. 

Experiments  have  demonstrated  that  not  all  mosquitoes  which  bite 
a  yellow-fever  patient  become  infected,  but  that  of  several  which  bite 
at  the  same  time  some  may  fail  either  to  get  the  parasite  or  to  allow  its 
later  development  in  their  body.  This  condition  is  similar  to  that  seen 
in  Anopheles,  with  regard  to  malaria,  j 

35 


546  APPENDIX 

How  long  do  infected  mosquitoes  remain  dangerous  to  the  non-immune 
community?  This  question  cannot  be  definitely  answered  at  present; 
there  is  good  presumptive  evidence  that  the  mosquito  may  harbor  the 
parasite  through  the  winter  and  be  enabled  to  transmit  in  the  spring  an 
infection  acquired  in  the  fall.  There  is  reason  to  believe  that  the  mos- 
quito, once  infective,  can  transmit  the  disease  at  any  time  during  the 
balance  of  its  life. 

With  regard  to  the  parasite  itself,  we  cannot  as  yet  say  much.  Number- 
less dissections  of  infected  insects  have  been  made,  and  serial  stained 
sections  have  been  prepared  at  various  periods.  The  latter  have  led  us 
to  expect  something  tangible  in  the  near  future,  though  until  now  nothing 
definite  has  been  discovered. . 

The  question  whether  other  genera  of  the  Culicidce  or  other  species 
of  Stegomyia  are  capable  of  transmitting  yellow  fever  is  still  open  for 
discussion.  It  is  best  not  to  forget  that  such  a  thing  is  possible,  though 
the  general  opinion  seems  to  be  that  yellow  fever  is  restricted  for  its 
propagation  to  the  genus  Stegomyia. 


INDEX. 


ABBF  condenser,  79 

Abbott's  bottle  for  collecting  water,  444 
Abscess,  various  producers  of,  329 
Achorion  Schoenleinii  (favus  fungus),  435 
Acid-fast  bacilli,  316 
Acids,  from  carbohydrates,  100 
effect  of,  on  bacteria,  111 
formation  of,  from  alcohol,  etc.,  101 
Actinomyces,  418 
isolation  of,  421 
occurrence  in  animals,  422 
Actinomycosis,  418 
Aerobic  bacteria,  41,  105 
Aerogenes  capsulatus,  bacillus,  380 
JSstivo-autumnal  parasite  of  malaria,  506 
Agar,  nutrient,  49 
Agglutination    of    bacteria    in    hanging 

drops,  82 
nature  of  substances  concerned  in, 

175 
relation  between  agglutinating  and 

bactericidal  powers,  184 
testing  of,  82,  175.      See  also  Indi- 
vidual bacteria. 

Agglutinins,  absorption  methods  for  dif- 
ferentiation of,  181 
characteristics  of,  176 
group,  177 
loss  of  power  of  bacteria  to  absorb, 

183 

specific  and  group,  178 
A<;glutinoids,  176 
Aggressins,  539 

Agramonte,  on  yellow  fever,  545 
Air,  bacteriological  examination  of,  448 
Alexines,   163,   170 
Amboceptor,  170 
Amoeba  coli,  477 

biological  characteristics  of,  479 
culture  of,  479 

examination  of  stools  for,  480 
Amoebae   in  diseases  other  than   dysen- 
tery, 481 
Amcebina,  470 
Anaerobic  bacteria,  41,  105 

culture  methods  for,   65 
Aniline  dyes,  69 

basic  and  acid,  70 
germicidal  properties  of,  115 
oil  as  mordant,  73 

Animals,  use  of,  for  diagnostic  and  test 
purposes,  135 


Anopheles  maculipennis,  509 
punctipennis,  509 
symptomatic,  389 
Anthrax  bacillus,  382 

biological  characters  of,  384 
growth  in  media,  385 
identification  of,  389 
infection,  how  caused,  388 

prophylaxis  against,  388 
morphology  of,  382 
non-spore-bearing  varieties,  386 
occurrence,  in  cattle  and  sheep, 

387 

in  man,  387 

pathogenesis,  386 

spore  formation,  386 

bacteriological  diagnosis  of,  389 

Antibacterial  sera,  testing  power  of,  159. 

170 

Antibodies,  in  general,  155,   162 
Antiseptic,  table  of  antiseptic  values,  116 

action,  107 
Antisera,  155,  162 
Antitoxins,  absorption  of,  161,  229 

Ehrlich's  theory  for  production  of, 

164 

elimination  of,  by  the  body,  161 
in  general,  155.    See  also  under  Diph- 
theria and  Tetanus, 
methods  of  administration  of,  161 
nature  of,  164 
other  theories  as  to  production  of, 

168 

production  of,  for  therapeutic  pur- 
poses, 159 

stability  of,  in  the  serum,  161 
Arnold's  steam  sterilizer,  48 
Arrhenius,  on  diphtheria  toxin,  214 
Arthrospores,  32 

development  of,  33 

Atkinson,  constitution  of  diphtheria  anti- 
toxin, 207 

Attenuation  of  virulence,  39 
Autoinfection,  152 
Autopsy  of  test  animals,  136 


B 


!  BACILLARY  dysentery,  253 
Bacilli,  acid-fast,  316 

general  characters  of,  27 
Bacillus.       See    also    under    individual 
names. 


548 


INDEX 


Bacillus  aerogenes  capsulatus,  380 

anthracis,  382 

symptomatic!,  389 

of  blue  pus,  374 

botulinus,  251 

of  bubonic  plague,  413    ' 

butter,  319 

coli  communis,  234 

diphtheria,  185 
-like,  195 

diplo-,  of  conjunctivitis.    See  Diplo- 
bacillus  of  Morax. 

of  Ducrey,  372 

of  dysentery,  253 

enteritidis,  248 

fsecalis  alcaligenes,  253 

of  Friedlander,  458 

of  glanders,  408 

of  green  pus,  374 

icteroides  (in  yellow  fever),  416 

of  influenza,  321 

of  Koch-Weeks,  327 

lepraB,  317 

of  leprosy,  317 

of  Lustgarten,  316 

of  malignant  cedema,  379 

mallei,'  408 

paracolon,  247 

paradysentery,  256 

paratyphoid,  247 

pneumo-,  of  Friedlander,  458 

proteus  vulgaris,  377 

pyocyaneus,  374 

of  smegma,  316 

of  soft  chancre,  372 

of  timothy  grass,  319 

of  tuberculosis,  288 

of  typhoid,  263 

of  whooping-cough,  325 
Bacteria,  adaptation  to  environment  of, 
38,  143,  144 

aerobic,  105 

in  air,  448 

anaerobic,  105 

attenuation  of,  39 

basic  forms  of,  25 

botanical  relationship  of,  24 

characteristics  of,  23 

chemical  composition  of,  39 

classification  of,  36 

cultivation  of,  56 

definition  of,  23 

destruction  of,  by  chemicals,  107 

effects  of,  87 

elimination  of,  from  the  body,  154, 
268,  284 

higher  forms  of,  24 

involution  forms,  34 

influence   of  one   species  upon   an- 
other, 42,  103 
of  reaction  of  media  on,  42 

local  effects  of,  144 

lower  forms  of,  24 

manner  in  which  they   excite   dis- 
ease, 145 


Bacteria,  motility  of,  86 
nitrification  by,  99 
nutrition  of,  40 
parasitic,  24 

products  of  the  growth  of,  87 
relation  of,  to  disease,  141 

to  other  micro-organisms,  24 
to  oxygen,  41 
to  temperature,  43 
reproduction  of,  31 
saprophytic,  24 
in  soil,  448 

spore  formation  of,  32 
staining  of,  69 
structure  of,  29 

symptoms  and  lesions  due  to,  prod- 
ucts of,  144 

toxin  production  by,  147 
in  water,  443 
Bacterial  ferments,  89 
proteins,  92 
species,  38 

permanence  of,  38 
Bactericidal  sera,  160 

properties  of  blood,   162 
substances,  origin  of,  176 
Bacteriology,  historical  sketch,  17 
Bacteroids  in  leguminous  plants,  99 
Beggiatoa,  40 
Beriberi,  442 

Biggs  on  antitoxin  treatment  of  diph- 
theria, 205 
Black-leg,  389 
Blastomycetes,  437 
Blood,  bactericidal  properties  of,  162 
examination  of,  in  malaria,  503 
infection  of,  153 
as  medium,  50 
-serum  coagulator,  51 
Blue  pus,  bacillus  of,  374 

disease,  517 

Bordet,  toxin  antitoxin  union,  95 
Botulinus  bacillus,  251 
Botulism,  251 

bacillus  of,  251 
Bouillon  media,  48 

nutrient,  48 

Bovine  tuberculosis,  308 
Bromine,  112 
Broth.     See  Bouillon. 

calcium,  352 

Bruce,  micrococcus  melitensis,  372 
Bubonic  plague,  413 

bacillus  of,  413 

biology  of,  415 
morphology  of,  413 
pathogenesis,  415 
diagnosis  of,  416 
immunity  against,  415 
Buchner,    alexine   theory   of    immunity, 

163 
method    for    an    aerobic    cultures, 

66 

Busse,  saccharomyces,  440 
Butter,  bacillus  of,  319 


INDEX 


549 


CALCIUM  broth.  352 

compounds  as  disinfectants,  111 
necessary  for  bacteria.  41 
Calkin's  study  on  smallpox.  523 
Capaldi  plate  medium,  280 
Capsules  of  bacteria,  30 

-taiiiin.ii  of.  74 
Carbohydrates,  action  of  bacteria  on,  100  ] 
Carbolic  acid  as  disinfectant,   115 

as  mordant,  73 
Carbon  dioxides,  production  of,  by  colon 

bacillus,  237 

Carroll,  yellow  fever,  442,  544 
Castellaiii,  on  absorption  of  agglutinins. 

181 
Cellulitis,    various   organisms   concerned 

in,  329 

Cercomonas  hominis,  495 
Cerebrospinal  meningitis,  360 
Chanciv.  soft,  bacillus  of,  372 
Charbon  symptomatique,  389 
Chemotaxis,  86 

Chloride  of  lime,  as  disinfectant,  112 
Chlorine,  as  disinfectant,  112 
Chloroform  as  disinfectant,  115 
Cholera.  Asiatic,  393 
diagnosis  of,  403 
inoculation  against,  402 
-red,  reaction,  396 
spread  of,  398 
spirillum,  393 

agglutination,  402 
allied  organisms  of,  404 
biology  of,  394 
identification  of,  403 
immunity  against,  402 
morphology  of,  394 
occurrence  outside  of  body,  397 
pathogenesis  of,  398 

:  ance  and  vitality  of,  397 
toxin  of,  401 
Ciliata.   17.', 
Cladothrix,  40,  431 
asteroides,  432 
liquefaciens,  431 
Classification  of  bacteria,  36 
Cnidosporidia,  518 

Cobbett  on  pseudodiphtheria  bacilli,  201 
Cocci,  characters  of,  26 

staphylococcus  pyogenes,  329 
streptococcus  pyogenes,  337 
Coccidiomorpha,  497 
Coccidium  bigeminum,  498 

cuniculi,  497 

Cold,  intense,  effect  of,  on  bacteria,  44 
Coley's  streptococcus  toxins,  342 
Collodion  sacs,  67 
Colon    bacillus,    action    on    nitrogenous 

compounds,  238 
behavior  toward  carbohvdrates, 

236 

biology  of,  235 
in  cystitis,  245 
in  diarrhoea,  243 


Colon  bacillus,  differential  diagnosis  from 

typhoid  bacillus,  285 
group  of,  234 
growth  of,  on  common  media, 

446. 

immunization  against,  451 
indol,  production  by,  238 
methods  of  isolation,  246 
morphology  of,  234 
occurrence  in  man  and  animals. 

241 

pathogenesis,  239 
in  peritonitis,  244 
in  sepsis,  243 

-typhoid  intermediates,  247 
Colonies,  characteristics  of,  59 
counting  of,  57 

study  of,  in  plate  cultures,  59 
various  forms  of,  59 
Comma  bacillus,  393 
Complement,  170 

deflection  of,  173 

Conradi  and  Drigalski,  medium,  280 
Copper  sulphate  as  disinfectant,  110 

capsule  stain,  74 
Councilman  on  smallpox,  521 
Counting  of  colonies,  57 
Cover-glass,    preparations,    how    made, 

68 

how  stained,  69 
how  to    render  slips  free  from. 

grease,  68 
thickness  of,  80 
Cowpox,  etiology  of,  522 

relation  to  smallpox,  521 
Crescent  bodies  in  malaria,  507 
Cresol,  115 

Culex,  in  malaria,  510 
Cultivation  of  bacteria,  56 
Culture,  anaerobic,  65 

media,  preparation  of,  47 

reaction  of,  52 
plate,  making  of,  56 
pure,  62 
Curtis,  saccharomyces,  440 


DECOLORIZING  of  stained  smears,  73 

Delhi  boil,  529 

Dengue,  442 

Diarrhoea,  relation  of  bacteria  in  milk  to, 

455 
Diphtheria,  antitoxin,  205 

nature  of,  207,  210-214 
persistence    of,    in    blood,  161, 

210 
production  of,   for  therapeutic 

purposes,  205 

result  of,  treatment  of,  205 
testing  of,  208,  210,  214,  215, 

543 

unit  of,  208 

use  of,   in   treatment   and  im- 
munization, 208 


550 


INDEX 


Diphtheria  bacillus,  185 

animal  inoculation  as  test  for 

virulence  of,  188 
biology  of,  188 
characteristic    appearances    of. 

187 

exudate  due  to,  contrasted  with 
that  due  to  other  bacteria, 
216 

agglutination  of,  210 
growth  on  agar,  190 

on  blood  serum,  190 
in  ascitic  bouillon,  192 
in  healthy  throats,  196 
human  inoculation  of,  186 
isolation    by    means    of    serum 

bouillon,  191 
of,     from    plate    cultures, 

191 
non-virulent    forms    of,     195, 

197 

pathogenesis,  192 
persistence  of,  in  throats,  196 

of  types  of,   199-204 
resistance  to  heat,  drying,  and 

chemicals,  189 
staining  of,  186 
virulent,    in    healthy    throats, 

196 

varieties  of,  200 
direct   microscopic    examination   of 

exudate  of,  220 

examination    of    cultures    for   diag- 
nosis, 219 
historical,  185 
-like  bacilli,  virulent  for  guinea-pigs. 

195 

mixed  infection  in,  198 
relation  of  bacteriology  to  diagnosis, 

215 
serum  for  eradicating  the  bacilli  of, 

from  the  throat,  209 
use  of,  in  eliminating  diphtheria  ! 

bacilli,  209 
susceptibility     to     and     immunity 

against,  204 

technique    of    bacteriological    diag- 
nosis, 217 
toxin,  193 

neutralizing    value    of    a    fatal 

dose,  210 
production  of,  in  culture  media, 

194 

relation  between  toxicity  and 
neutralizing  value  of,  210, 
211 

union  with  antitoxin,   207 
toxoid,  210 
toxon,  213 
transmission  of,  204 
value   of    cultures   in   diagnosis   of, 

Diphtheritic  characteristic  appearance  of, 

216 

inflammations,  location  of,  196 
Diplobacillus  of  conjunctivitis,  543 


Diplococcus   intracellularis  meningitidis, 

360 
of   pneumonia,    349.     See   Pneumo- 

coccus. 

Disinfectants,  gaseous,  111 
Disinfection,  107 
by  heat,  45 

practical,   of  house,   person,  instru- 
ments, and  food,  117 
Dissemination  of  disease,  151 
Drigalski  and  Conradi,  medium,  280 
Drying,  effects  of,  on  bacteria,  104 
Ducrey,  bacillus  of  soft  chancre,  372 
Dunham's  peptone  solution,  49 
Duval,  dysentery  bacillus,  257 
Dysentery,  amoebic,  477 
baciflary,  253 
bacillus,  253 

agglutination    characteristics, 

259 

biology  of,  254 
mannite    fermenting    varieties, 

257 

morphology  of,  254 
pathogenesis  of,  255 
relation  to  paradysentery 

bacilli,  256 
types  of,  261 
historical  notes  of,  254 
pathology  of,  253 


EBERTH,  bacillus  of  typhoid,  263 
Ehrlich  on  diphtheria  toxin,  212 

on   the   nature   of   toxins,    94,   164, 
210 

partial  saturation,  method  of,  212 

side-chain  theory  of,  164 

standardization  of  diphtheria  anti- 
toxin, 210 

Electricity,  influence  on  bacteria,  102 
Elimination  of  bacteria  through  milk,  153 
through  skin  and  mucous  mem- 
brane, 154 

through  urine,  268-284 
Enantobiosis,  103 
Endospores,  32 
Endotoxins,  95 
Entamceba  coli,  477 

hystolytica,  478 

Enteric  fever,  441.     See  Typhoid  fever. 
Enzymes,  88 
Epitoxoids,  210 
Erlenmeyer's  flask,  54 
Erysipelas,  streptococci  in,  337 
Estivo-autumnal  parasite  of  malaria,  506 
Examination  of  air,  448 

collection  of  material  for,   138 

of  feces  and  urine  for  typhoid  bacilli, 
278 

of  ice,  543 

of  unstained  bacteria,  81 

of  water,  443 
Eye-piece,  77 


551 


1  A* n.i  A  1 1\  i:  aerobic  and  anaerobic  bac- 
teria, 10(3 

Faecalis  alealiiz;cnes,  bacillus,   253 
Fats,  decomposition  of,  98 
Feces.  disinfection  of,  119 

examination  of,  for  typhoid  bacilli, 

278 
Fermentation  by  bacteria,  88 

tube,  101 
Ferments,  characteristics  of,  89 

ilia  static,  89 

inverting,  89 

organi/.ed  and  unorganized,  88 

proteolytic,  89 

rennin-like,  90 
Field,     .studies    in     scarlet     fever    and 

measles,  528 
Film  preparations,  68 
Filtration  of  water,  449 
Finkler  and  Prior,  spirillum  of,  405 
Fission  fungi,  23 
Fixation  of  smears,  69 
Flagella.  30 

staining  of,  76 
Flagellata,  472 

life  cycle  of,  473 
Flexner,  dysentery  bacillus,  256 
Flies,  relation  to  trypanosomiasis,  488 
Focusing,  80 
Formaldehyde  as  disinfectant,  112 

Wilson's  rapid  generator,   127 
Fractional  sterilization,  56 
Friedlander,  bacillus  of,  358 
Fungi,  fission,  23 

pathogenic  varieties,  433 
Fungus  of  favus,  435 

of  pityriasis,  436 

ray  (actinomyces),   418 

of  ringworm,  433 

of  thrush  (soor),  437 


GABBETT'S  solution,  312 
Gametes  in  malaria,  505 
Gartner,    bacillus  enteritidis,   251 
Gas  production  by  bacteria,  100 

test  for,  101 
Gelatin  media,  49 
(termination  of  spores,  34 
Gessard,  bacillus  pyocyaneus,  374 
Giemsa,  stain,  493 
Glanders  bacillus,  408 

i-olation  of,  411 
morphology,  408 
pathogenesis,  409 
diagnosis  of,  411 
immunity  against,  411 
test  for  (mallein),  411 
Globulin,  relation  of  serum  globulin  to 
diphtheria  antitoxin,  207  V 

Glossina  palpatis  in  relation  to  trypano- 
somiasis, 488 


Glucose  bouillon,  48 
Glycerin  agar,  49 
Goldhorn  stain,  511 
Gonococcus,  bacteria  resembling,  372 
culture  media  for,  368 
diseases  excited  by,  369 
in  endocarditis,  369 
occurrence  of,  371 
staining  reactions,  367 
Gonorrhoea,  bacteriological  diagnosis  of, 

370 

Gram's  stain,  74,  367 
Granules,  metachromatic,  34 
Grass  bacillus,  319 
Green  pus,  bacillus  of,  374 
Group  agglutinins,  177 

reaction,  174 
Gruber-Widal  reaction,  270 

persistence  of  reaction,  277 
relation  of,  to  typhoid,  277 
use  of  dead  cultures  for,  274 
of  dried  blood  for,  273 
of  serum  for,  274 
Guarni£re,  vaccine  bodies,  522 


H^MOLYSINS,  Ehrlich's  studies  on,  540 

Haemosporidia,  500 

Haffkine's    preventive    inoculations    for 

cholera,  402 
for  plague,  416 
Hands,  disinfection  of,   132 
Hanging  drop  for  study  of  bacteria,  44 
Hansen,  bacillus  of  leprosy,  317 
Haptophore  group,  164 
Hauser,  bacillus  proteus  vulgaris,  377 
Heat,  effect  of  dry,  on  bacteria,  44 

of  moist/ on  bacteria,  45 
Hiss,  capsule  stain,  74 

media  for  typhoid  bacillus,  279 

serum  media,  52 

Historical  sketch  of  bacteriology,  17 
Hollow  slide,  81 
Hydrogen  peroxide,  112 

production  of,  by  colon  bacillus,  237 
Hydrophobia,  530.     See  Rabies. 


ICE,  bacteria  in,  44,  543 
Icteroides,  bacillus,  416 
Immune  body,  170 
Immunity,  active,  157 

duration  of,  161 

passive,  158 

specific,  157 

theories  of,  162,  168,  171,  172 
Impetigo  contagiosa,  442 
Incubators,  64 
Indol,  238,  396 

test  for,  98 
Infection  of  blood,  153 

influence  of  quantity  in,  146 


552 


INDEX 


Infection,  mixed,  148 

modes  of  entrance,  149 

spread  of,  151 

protection    afforded    by    skin    and 

mucous  membranes,  149 
Inflammation  due  to  bacteria,  144 
Influence  of  one  species  upon  growth  of 

another,  42,  103 
Influenza  bacillus,  321 

agglutination  of,  326 

cultivation,  322 

distribution  in  the  body,   323, 

324 

immunity  to,  323 
morphology  of,  322 
pathogenesis,  323 
resistance,  323 
staining  characteristics,  322 
in  tuberculosis,  324 
Infusion,  meat,  47 
Instruments,  dressings,  etc.,  disinfection 

of,  131 

Interbody,  170 
Intestines,   development  of  bacteria  in, 

150 

Inulin  in  serum  media,  52 
Invisible  micro-organisms,  442 
Involution  forms  of  bacteria,  34 
Iodine,  74 
lodoform,  115 


JAPANESE  worm,  440 


KALA-AZAR,  diagnosis  of,  529 
Leishman  bodies  -in,  529 
Kitasato,  222 
Koch,  Robert,  22 

cholera  spirillum,  393 
tubercle  bacillus,  288 
-Weeks  bacillus  of  conjunctivitis,  327 
differential,     diagnosis 

of,  328 

morphology  of,  327 
Kruse,   dysentery  bacillus,  256 


LACTOSE  bouillon,  48 

Lamblia  intestinalis,  496 

Laveran,  plasmodium  of  malaria,  500 

Leeuwenhoeck,  first  microscope  of,  18 

Leishman  bodies,  529 

Leprosy  bacillus,  317 

differential  diagnosis  of,  319 

location  of,  in  tissues,  318 

morphology  of,  317 

pathogenesis  of,  318 
Leukocytes,  production  of  exudates  rich 

in,  157 
part  played  by,  in  immunity,  172 


Light,  production  of,  by  bacteria,  86 
influence  of,  on  bacteria,  102 

Litmus,  as  indicator,  53 
media,  50 

Loeffler's  alkaline  solution  of  methylene 

blue  for  staining  diphtheria,  73 
blood  serum,  51 

Lustgarten's  bacillus,  316.     See  Smegma. 


MACNEAL,  cultures  of  trypanosomes,  484 

Macrogametes  of  malaria,  505 

Mai  de  Caderas,  487 

Malaria,     500.       See    also    Plasmodium 

malarise. 

infection,  how  acquired,  509,  513 
-like  organisms  in  other  animals,  513 
mosquitoes  in  relation  to,  509 
technique  of  blood  examination  in, 

503 
Malignant  cedema,  bacillus  of,  379 

pustule,  387 
Mallei,  bacillus,  408 
Mallein,  test  for  glanders,  411 
Mallory,  studies  on  scarlet  fever,  527 
Malta  fever,  372 
Mannite  fermenting  dysentery  bacilli,257, 

372 

in  media,  48,  49 
Marble  broth,  352 
Marmorek,  media,  52 

serum  for  tuberculosis,  308 

antistreptococcus,  344 
Material  for  bacteriological  examination, 
procuring  of,  from  those  suffering  from 
disease,  138 

Measles,  Field's  studies  on,  441 
Meat  infusion,  47 

poisoning,  252 

Media,  preparation  and  sterilization  of,  47 
reaction  of,  52 
various  kinds  of,  48 
Melitensis,  micrococcus,  372 
Meningitis,  bacteriological   diagnosis   of, 

363 

various  organisms  exciting,  364 
Meningococcus,  360 
agglutination,  363 
biological  characteristics,  361 
pathogenesis,  362 
presence  in  nares  of  both  sick  and 

healthy  persons,  363 
in  blood,  363 

Mercury  bichloride  as  a  disinfectant,  109 
Mesophilic  bacteria,  43 
Metachromatic  granules,  34 
Metchnikoff  on  immunity,  163 

spirillum  of,  406 
Meyer  and   Ransom   on   tetanus  toxin, 

229 
Micrococcus  catarrhalis,  365 

gonorrhoea,  366.     See  Gonococcus. 
intracellularis,    360.      See   Meningo- 
coccus. 


INDEX 


553 


Micrococcus  tanoeolatUB,  349.    SeePivn- 

mococcus. 
melitensis,  372 
tetrairenus  335 
Microgametes  ot  malaria,  505 
Microscope,  different   parts  of,  77 
Microscopic    examination    of    unstained 

bacteria,  81 
Microsporidia,  518 

Miiiula,   classification  of  bacteria,  36 
Milk  bacteriology  of,  in  relation  to  dis-  ; 

153 

as  culture  medium,  49 
heated  vs.  raw,  in  feeding,  456 
influence  of  cleanliness  on,  465 

of    tcmperatute    on    growth    of 

bacteria  in,  462 
number  of  bacteria  in,  463 
pasteurization  of,  134 
pathogenic  properties  of,  455 
Merili/ution  of,  132 
time  required  for  multiplication  of 

bacteria  in,  463 
transmission    of    disease    through, 

467 

Milzbrand,  382.     See  Anthrax. 
Moeller's  method  of  staining  spores,  75 
Mixijuitoes    as    agents    of    infection    in 

malaria,  509 
in  yellow  fever,  442,  544 
Morax,     diplobacillus   of    conjunctivitis, 

543 

Mordants,  73 
M utility  of  bacteria,  86 
Mucous  membranes,  ability  of  bacteria 

to  penetrate,  149 
Mumps,  442 
Mycelium,  21 
Mycetozoa,  471 
Myxosporidia,  518 


N 


NEGRI  bodies  in  rabies,  531 
Neisser  gonococcus,  366 

stain  for  diphtheria  bacilli,  187 

and  AYechsberg  phenomenon,  17.'-} 
Neutral  red,  50 
Nitrate  bouillon,  50 
Nitrification,  99 

Nitrites  and  nitrates  produced  b\    bac- 
teria, 99 

Nitroso-indol  reaction,  98 
Noma.  442 
No-i-Mia  bombycis,  519 

lophii.  .")!'.) 

Now  and  MacNeal,  cultures  of  trypano- 
somes,  484 

method   of    making    anaerobic    cul- 
tures, 66 


OlL-IMMKHsloN    lens,   79 

Opsonines,  172 


PARACOLON  bacilli.  247 

Paradysentery  bacilli,  247 
Parasites,  strict,  1  1 1 
Paratyphoid  bacilli,  248 
Pasteur,  early  investigations,  20 
flask,  54 

treatment  of  rabies,  533 
Pasteurization,  46 
Pemphigus  neonatorum,  442 
Peptone  solution,  Dunham's,  49 
\\-<\   (bubonic  plague),  413 
Petri  dish,  57 

Petrusky's  litmus-whey,  50 
Pfeiffer,  influenza  bacillus,  321 

micrococcus  catarrhalis,  365 
Pfeiffer's  phenomenon,  271 
Phagocytosis,  163 
Phenolphthalein  as  indicator,  53 
Pigment  production  by  bacteria,  90 
Piro plasma  bigeminum,  5! 4 
Pityriasis  versi color,  436 
Plague,  bubonic,  413 
Plasmodium  of  malaria,  502 

aestivo-autumnal  parasite,  506 
classification,  502 
examination  of  blood  for,  503 
quartan  parasite,  508 
staining  methods  for,  501 
tertian  parasite,  502 
prsecox,  502 
vivax,  502 
Plate  cultures,  study  of  colonies  in,  59 

technique  of  making,  56 
Plenciz,  Antoiiius,  18 
Pneumobacillus  of  Friedlander,  358 
Pneumococcus,  349 

agglutination  reaction,  358 
biological  characteristics,  350 
effects  of  drying  and  sunlight  on, 

352 

immunity  to  infection  by,  357 
mucosus*  358 

occurrence  in  man  in  health,  354 
pathogenesis,  353 
presence  in  lobar  and  bronchopneu- 

monia,  354 
varieties  of,  357 
Polymastigina,  496 
Potatoes  as  culture  medium,  50 
Precipitins,  176 

1 're-sure,  influence  of,  on  bacteria,  102 
Prior,  spirillum  of  Finkler  and,  405 
Proteins,  bacterial,  92 
Proteus,  bacillus,  377 
Prototoxoid,  211 
Protozoa,  469 

classification,  470 
Protozoan-like  bodies  in  smallpox  and 

allied  diseases,  521 
Pseudodiphtheria  bacilli,  197 
Pseudonienibranous  inflammations  due  to 
bacteria  other  than  diphtheria  bacilli, 
198 
Pseudotubercnlosis,  streptothrix  in,  423 


36 


554 


INDEX 


Pseudoworm,  440 
Psittacosis  bacillus,  248 
Psychrophilic  bacteria,  43 
Ptomaines,  91 
Pure  cultures,  62 

in  tubed  media,  63 
Pustule,  malignant,  387 
Putrefaction,  98 
Pyocyaneus,  bacillus,  374 
Pyocyanin,  92 
Pyogenic  cocci,  329 
Pyrosoma,  514 


QUARTAN  parasite  of  malaria,  508 
Quarter  evil,  389 


RABIES,  441,  530 

cauterization  of  wounds  in,  537 
Negri  bodies  in,  531 
Pasteur's  treatment,  533 
preventive  inoculation  against,  533 
Radium,  influence  on  bacteria,  103 
Rauschbrand,  389.     See  Symptomatic 

anthrax. 

Ray  fungus,  418.     See  Actinomyces. 
Reaction  of  media,  correction  of,  52 
Receptors,  164 
Reduction  processes,  effect  by  bacteria, 

97 

Reed,  yellow  fever,  442 
Relation  between  agglutinating  and  bac- 
tericidal power,  181 
Relapsing  fever,  spirillum  of,  490 
Ross,  relation  of  mosquitoes  to  malaria, 
500 


SACCHAROMYCES,  439 
Busse,  440 
neoformans,  441 
subcutaneus  tumefaciens,  440 
Sanarelli,  bacillus  icteroides,  416 
Sanfelice,  441 

Saprophytes,  facultative,  144 
Sausage  poisoning,  251 
Scarlet  fever,  Field's  studies  on,  528 

protozoan-like    bodies   in,    441, 

527 

streptococci  in,  441 

Schaudinn,  spirochsete   pallida  in  syph- 
ilis, 492 

Srhizomycetes,  23 

Schizosaccharomyces  octosporus,  439 
Srliocnlcin,  achorion  of,  435 
Scurvy,  442 

Sections,  preparation  of,  76 
Septicaemia,  various  organisms  concerned 

in,  329 

Sera,  antitoxic,  162 
k-irtorioidal,  170 


Serum,  alkaline  blood,  51 

antimeningitis,  363 

antipneumococcus,  358 

antistreptococcus,  344 

antityphoid,  270 

collection  of,  for  diagnostic  purposes, 
140 

diagnosis,   270.      See  Gruber-Widal 
reaction. 

limit  of  curative  power,  159 

Leoffler's  blood,  51 

media,  51 

testing  of  protective  power  of,  159 
Sewage,  disposal  of,  451 
Shiga,  dysentery  bacillus,  254 
Side-chain  theory  of  Ehrlich,  164 
Silkworm  disease,  519 
Skin,  ability  of  bacteria  to  penetrate,  149 

disinfection  of,  Fiirbinger's  method, 

132 

Sleeping  sickness,  482,  488 
Smallpox,  441 

protozoan  bodies  in  smallpox  and 

allied  diseases,  521 
Smears,  staining  of,  69 
Smegma  bacillus,  316 

differential  diagnosis,  316 
morphology,  316 
relation  to  syphilis,  316 
staining  characteristics,  316 
Soil,  bacteria  in,  448 
Soor,  fungus  of,  437 
Spallanzani,  19 

Species,  influence  of  one  upon  growth  of 
another,  42,  103 

permanence  of,  38 
Specific  agglutinins,  178 
Specificity  of  agglutinins,  176 
Spirilla,  general  characteristics  of,  28 

allied  to  cholera,  404 
Spirillum  of  Asiatic  cholera,   393.     See 
Cholera. 

of  Finkler  and  Prior,  405 

of  Metchnikoff,  406 

of  Obermeier,  490 

of  relapsing  fever,  490 
Spirochsete  of  Obermeier,  490 

pallida,  492 

of  Schaudinn,  492 
Sporagenous  granules,  42 
Spores,  32 

effect  of  heat  on,  44 

germination  of,  34 

resistance  to  heat,  44 

staining  of,  75 
Sporozoa,  472 

Spotted  fever,  517.    See  also  Meningitis. 
Sputum,  disinfection  of,  120 

methods  of  examination  for  tubercle 

bacilli  in,  310 
Staining  bacteria,  70 

principles  underlying,  70 
Stains,  Gabbett,  312 

Giemsa,  493 

Goldhorn,  511 

(Irani,  74 


INDEX 


555 


Stains,  Hi-.  71 
Jenner,  511 
Koch-Ehrlich.  73 
Loeffler's,  73,  76 

Moeller,  T:> 
Neisser,  187 
Nocht-Romanowsky,  511 

Welch.    74 

Ziehl-Xeelsen,  73 

Staphylococcus  epidermidis  albus,  334 
pyogenes,  329 

occurrence  in  man,  333 
pathogenesis,  331 
varieties,  334 

Stegomyia    fasciata    (yellow-fever    mos- 
quito), 544 
Sterilization,  107 

fractional,  45,  56 
of  milk,  132 
Sterilizer,  dry  heat,  55 
Stitch  abscess,  335 

Stomach,  as  protection  against  bacterial 
invasion,  150 


'is  iii  iiuu. 

natural  infection,  225 
toxin,  absorption,  230 

action  of,  in  body,  225 
neutralization  of,  228 
of,  in  body,  231 
presence  in  blood,  227 
union  with  antitoxin,  229 
treatment  with  antitoxin,  231 
-  fever,  parasite  of,  613 

prophylaxis,  515 
Thermophilic  bacteria,  43 
Thermo-regulator,  64 
Thrush  fungus,  437 
Ticks,  Boophilus  bovis,  515 
Ixoides  redivius,  515 
in  relation  to  disease,  515 
Timothy  grass  bacillus,  319 
Tinea  barbae,  433 
circinata,  433 
sycosis,  433 
tonsurans,  433 
Tissue,  examination  of  bacteria  in,  76 


Streptococci,  general  characteristics,  337    Titration  of  culture  media,  53 


Streptococcus  mucosus  capsulatus,  358. 

See  Pneumococcus  mucosus. 
pyogenes,  337 

cultivation,  339 
identification,  348 
immunization  against,  343 
pathogenesis,  340 
toxic  substances  produced  by, 

343 

Streptothrix.  24 
biology,  428 
infection  by.  423 
in  pseudotuberculosis,  423 
spore  formation  by,  429 
Strong,  dysentery  bacillus,  256 
Sulphur  dioxide  gas  in  house  disinfection. 


130 

Sulphuretted    hydrogen,    production 

97 

Sunlight,  influence  on  bacteria,  103 
Symbiosis,  103 
Symptomatic  anthrax.  389 
Syntoxoids.  211 
Syphilis.    \  \ 

spirocha-te  pallida  in,  492 
Lustgarten's  bacillus  in,  316 


Toxins,  Ehrlich's  theory  as  to  the  nature 

of,  94,  164 
Toxoids,  165 
Toxon,  213 
Toxophore  group,  1<>4 
Trichomonas  vaginalis,  496 
Trichophyton  megalosporon,  433 

microsporon,  433 
Tropical    malaria,     506.       See    JSstivo- 

autumnal  malaria. 
Trypanosoma,  482 

Brucei,  486 

Evansi,  485 

Lewisi,  482 

in  man,  487 

Theileri,  487 


TKMPKKATUKE,  effect  of,  on  bacteria.  44 
Tetanus,  antitoxin.  229 

bacillus,  biology  of,  222 
duration  ot'  life,  225 
in  intestines.  225 
morphology,  222 
non-virulent    type.   232 
occurrence  in  soil,  225 

221 
223 

staining  M,  222 
differential  diacn<»<is.  232 


Trypanosomiasis,  487 
of,  method  of  examination  for,  489 

serum  therapy  in,  490 
symptoms,  488 
Tubed  cultures,  63 
Tube-length,  80 

Tubercle  bacillus,  agglutination,  302 
avian,  308 
biology,  289 
bovine,  308 
cultivation,  290 
diagnosis  by  animal  inoculation, 

315 

discovery,  288 
distribution,  288 
examination  of  material  for,  310 
human,  308 
immunization,  301 
method    of    making    pure    cul- 
tures. 292 
morphology.  288 
pathogenesis,  294 
poisons,  295 

ining  peculiarities.  289 

in  tissues.  :^l.~) 
viability.  301 


556 


INDEX 


Tuberculin,-  diagnostic  use  of,  305 
new,  304 
old,  303 
Tuberculosis,  281 

mixed  infection,  301 
mode  of  infection,  297 
prophylaxis,  308 
serum  treatment  of,  308 
Tuttle,  on  streptothrix,  424 
Typhoid,  441 

bacillus,  agglutination  of,  270 

biological  characteristics,  264 

cultures,  265 

distribution  in  human  subject, 

267 
duration  of  life  outside  the  body, 

268 
elimination    of,    through    urine 

and  feces,  268,  283 
in  feces,  283 
identification,  285 
isolation  of,  278 

Capaldi  method,  280 
Hiss'  method,  279 
Drigalski     and     Conradi 

method,  280 
occurrence  in  water,  oysters,  and 

milk,  268,  269 
unusual  localization,  267 
in  urine,  268 
-colon  intermediates,  247 
communicability,  269 
diagnosis  by  means  of  serum  test, 

270 

due  to  infected  milk,  468 
Gruber-Widal  reaction  in,  270 
immunization  against,  270 
Typhus  fever,  441 
Tyrotoxicon,  91 


ULTRAMICROSCOPIC  examinations,  542 
Urea,  fermentation  of,  91 
Urine,  bacteria  eliminated  through,  268 
typhoid  bacilli  in  284,  268 


VACCINATION,    immunity    conferred   by, 

o22 
Vaccine,  bacteria  in,  526 

bodies,  522 

durability  of,  526 

preparation  of,  524 
Variola,  etiology  of,  522 
Vibrio  Berolinensis,  404 

of  cholera,  393 

Danubicus,  404 

Metchnikovi,  406 

Virulence,   variation  in  degree  of,   pos- 
sessed by  bacteria,  146 


WASSERMANN,  diphtheria  serum,  209 

on  formation  of  antitoxin,  167 
Water,  bacilli   of    colon   group   in,    443, 

445 

bacteriological  examination  of,  443 
interpretation     of    results, 

446 
contamination   and   purification   of, 

449 

proteus  bacilli  in,  447 
purification  of,  for  domestic  purposes, 

450 

on  large  scale,  449 
streptococci  in,  447 
typhoid  bacilli  in,  447 
Weeks'  bacillus,  327.     See  Koch-Weeks. 
Weichselbaum,  meningococcus,  360 
Weigert,  Carl,  22 
Weigert's  law,  167 
Welch,    bacillus    aerogenes    capsulatus,. 

380 

capsule  stain,  74 
staphylococcus    epidermidis    albus, 

334 
Whooping-cough,  agglutinins  of,  325 

bacilli  found  in,  325 
Widal  reaction,  270.    See  Gruber-Widal. 
Williams,  study  on  diphtheria,  202 

pneumococcus  mucosus,  358 
Wilson  on  agglutination,  82 

apparatus  for  formaldehyde  disinfec- 
tion, 127 

method  for  anaerobic  cultures,  66 
Wollstein    on    whooping-cough    bacilli, 

325 

Wool-sorters'  disease,  382 
Worm,  Japanese  or  pseudo-,  440 
Wright  on  actinomyces,  421 

inoculation  against  typhoid,   270 
on  kala-azar,  529 
opsonine,  172 


X-RAYS,  influence  of,  on  bacteria,  103 


YEASTS,  437 

culture,  438 

wild,  438 

Yellow  fever,  bacillus  icteroides,  416 
mosquitoes  in,   442,   544 


ZIEHL'S  carbol-fuchsin  solution  for  tuber- 
cle bacilli,  73 
Zymophore  group,  165 


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